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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3167-3215
REVIEW ARTICLE
Deconstructing a Disease: RAR , Its Fusion Partners, and Their
Roles in the Pathogenesis of Acute Promyelocytic Leukemia
By
Ari Melnick and
Jonathan D. Licht
From the Derald H. Ruttenberg Cancer Center and Department of
Medicine, Mount Sinai School of Medicine, New York, NY.
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INTRODUCTION |
IN THE LATE 1980s and early 1990s, the
elucidation of the molecular basis of acute promyelocytic leukemia
(APL) emerged as a paradigm for the connection between the bench and
bedside. At that time, it became apparent that APL was, among the forms of acute myeloid leukemia, uniquely sensitive to all-trans
retinoic acid (ATRA)1,2 and clinical trials indicated
that ATRA induced complete remissions by differentiation and eventual
elimination of the malignant clone (reviewed
previously3-8). In 1991, it was discovered that the
consistent chromosomal translocation of APL, t(15:17),9
fused the retinoic acid receptor  (RAR) gene to the promyelocytic
leukemia (PML) gene on chromosome 15, yielding the fusion protein
PML-RAR.10-15 These data suggested that disruption of
RAR function was the major cause of APL. According to this line of
reasoning, retinoic acid in pharmacological doses could then overcome
this pathology, leading to in vivo differentiation and clinical
remission. Although this hypothesis is essentially correct, 7 years of
intense investigation of the APL model have begun to uncover a more
complicated picture.
APL is now associated with four different gene rearrangements, fusing
RAR to the PML, promyelocytic leukemia zinc finger (PLZF),
nucleophosmin (NPM), or nuclear matrix associated (NuMA) genes
(Fig 1), leading to the formation of reciprocal
fusion proteins (N-RAR and RAR-N). This again highlights the
importance of retinoid metabolism, but also suggests that partner genes
with RAR could also play important roles. In this review, we will
deconstruct the APL problem by evaluating the role of RAR in normal
and neoplastic myeloid development. We will examine each of the genes
fused to the RAR in APL, searching for similarities and differences
among the four partner proteins that may explain the distinct clinical outcome some patients with variant forms of APL. Finally, we will reconstruct the disease of APL and examine the leukemogenic functions of the RAR fusion proteins in cell culture models, animal models, and patients. We will also examine how the recent explosion of knowledge in APL has led to the development of new therapeutic agents
such as arsenic trioxide16,17 and sodium butyrate.
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| Fig 1.
The four chromosomal translocations associated with APL
result in fusion proteins in which the B through F domains of RAR ,
including the DNA binding and ligand binding domains of protein, are
linked C-terminal to four different nuclear proteins containing
self-association domains. The t(11;17) APL syndrome linking PLZF and
RAR is unique among these forms of APL in its resistance to
differentiation therapy with ATRA or conventional chemotherapy.
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THE RETINOIC ACID RECEPTOR |
Retinoids may be key for myeloid differentiation. Vitamin A-deficient
mice and humans were noted to have defects in
hematopoiesis18,19 and retinoids can preferentially
stimulate granulopoiesis.20,21 In the early 1980s, it was
noted that retinoic acid (ATRA) could induce differentiation of myeloid
cell lines such as HL6022 and of primary cells from
patients with APL.23 The cloning of the RARs and other
members of the nuclear receptor superfamily24-26 allowed
for further detailed studies into the mechanism of action of ATRA.
Among the genes encoding RARs (reviewed previously24-26), RAR is identified with myeloid development.27-29
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TRANSCRIPTIONAL FUNCTION OF RAR |
RAR structure parallels that of other nuclear receptors and is
divided into 6 evolutionarily conserved domains (A through F; Fig 2).
The most highly conserved domain among nuclear receptors and retinoid
receptors is the C domain, which contains two
C2C2 zinc finger motifs (reviewed in
Chambon30). Through this domain, RAR binds to retinoic
acid response elements (RARE) located in the promoters of many genes,
including those of RAR,31,32 RAR ,33,34
and RAR .35 RAREs consist of a direct repeat
(A/G)G(G/T)TCA separated by 2 or 5 nucleotides. RAR binds as a
heterodimer to this site along with the related retinoid X receptor
protein (RXR).36-38 Heterodimerization is mediated both by
the DNA binding and ligand binding domain of RAR.39,40
RAR is a ligand-dependent transcription factor stimulated by ATRA,
whereas its partner, RXR, responds to ATRA or 9-cis retinoic
acid.41 RAR and other nuclear receptors contain two
domains, AF-1 (A/B domains) and AF2 (E domain), which can cooperate to
activate transcription.42 AF-1, contained within the
N-terminal A/B domain, is a ligand-independent transcriptional activation domain that works in a promoter context-dependent
manner.42,43 Through alternative promoter usage, the RAR
protein can have two different A domains (A1 or A2). The C-terminal E
domain of RAR contains the AF2 ligand-dependent transcriptional
activation domain as well as a dimerization interface for
RXR.39,40,43
RARs modulate transcription through interaction with cofactors. The
AF-2 domain of the protein associates with corepressor molecules in the
absence of ligand. These corepressors, N-CoR and SMRT,44,45
were recently shown to be part of a multiprotein repressor complex also
containing the Sin3A corepressor and histone deacetylases (Pazin and
Kadonaga46 and references therein). This suggests that RARs
may silence certain promoters by alterations in chromatin
configuration. Structural studies of RXR and RAR indicate that,
in the presence of ligand, the AF2 changes its conformation, making new
residues available30,47-49 to bind to coactivator proteins.
Nuclear receptor coactivators include TIF150,51 related to
the PML protein associated with t(15;17)-associated APL (see below),
Trip1/sug1,52 Tif2,53 ACTR,54
Src-1,55 TAFII135,56 and
CBP.57-59 The functions of these coactivators are beginning
to be elucidated. TIF150 interacts with the TATA binding
protein (TBP), TBP-associated factors (TAFs),60 and the
basal factor TFIIE.61 Murine and yeast Trip1/sug1 have DNA helicase activity,62 which could unwind DNA, whereas the
CBP and ACTR cofactors have histone acetylase activity and associate with P/CAF, another histone acetylase.54,63-66 It is
believed that histone acetylation leads to alterations in the
conformation of chromatin and stimulation of gene
transcription.67 Hence, the ligand-activated RAR can
best be imagined as a multiprotein complex bound to DNA in association
with RXR and a number of coactivator proteins. The ligand bound complex
might then stimulate transcription though interaction with basal
factors, alteration of chromatin, and unwinding of DNA.
The use of synthetic ligands specific for RAR and RXR indicate that
RAR/RXR complexes that stimulate gene transcription are responsible for
the pro-differentiation effect of ATRA,68-71 whereas RXR/RXR complexes cannot induce differentiation of APL cells. RARs can
also repress transcription through cross-talk with other transcriptional activators, including the AP1 family of activator proteins,72 probably due to competition for limiting
coactivators such as CBP.73-75 However, the most important
action of RAR in myeloid differentiation is its ability to activate
transcription through RAREs, because artificial ligands that inhibit
AP1 activity but fail to stimulate RARE-mediated transcription fail to
induce myeloid differentiation.68,76 Which key genes are
stimulated to affect myeloid differentiation remain to be determined.
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RAR AND MYELOID DIFFERENTIATION |
The importance of RAR in myeloid differentiation was underscored
when Collins et al77-79 developed a HL60 cell line
resistant to differentiation by ATRA. This cell line harbored a
dominant negative mutant RAR with a truncation within the C-terminal
AF-2 domain. Differentiation of these cells under the influence of ATRA
was restored by infection with a retrovirus expressing wild-type RAR, RAR, or RAR.77,80 Furthermore, RXR
expression in the resistant cells restored myeloid differentiation,
suggesting that the mutant receptor may have acted in a dominant
negative mode by heterodimerizing with wild-type RXR and forming an
inactive transcriptional complex. Overexpression of RXR overcame this
block, perhaps by recruiting other isoforms of RAR to mediate the
transcriptional response required for differentiation.
RAR may help program normal hematopoietic development. Erythroid
induction of multipotent FDCP mixA4 cells by erythropoietin was
correlated with complete downregulation of RAR expression, whereas
myeloid differentiation induced by granulocyte colony-stimulating factor (G-CSF) was correlated with upregulation of RAR, particularly the RAR2 isoform.81 Introduction of an RAR mutant,
with a deletion in the ligand-binding domain, into a multipotential
hematopoietic cell line resulted in a switch in cell fate from the
granulocyte/monocyte to the mast cell lineage.82
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated
myeloid differentiation of these cells was blocked at the promyelocyte
stage, an effect that could be overcome by high doses of
ATRA.83 Although truncation of the RAR within the ligand
binding domain has a profound effect on myeloid differentiation, this
type of mutation was not identified in a series of 118 specimens of
human cancer, including a number of fresh APL specimens.84
In leukemia, the RAR gene is only disrupted by the formation of
chromsomal translocations yielding fusion genes (see below). The notion
that the dominant negative RAR functions by sequestration of RXR
into inactive complexes was supported by the finding that
overexpression of wild-type RAR in murine bone marrow
cultures85 led to the accumulation of promyelocytic
colonies. Upon addition of ATRA, the RAR-expressing marrow colonies
consisted mainly of more differentiated granulocytes. Hence,
overexpression of wild-type RAR, C-terminal truncated forms of
RAR and fusion proteins consisting of partners fused to the
N-terminus of RAR (eg, PML-RAR; see below) can all lead to the
blockade of myeloid differentiation at the promyelocyte stage when
cells are grown at physiological levels (~10 8
mol/L) of ATRA. Only pharmacological levels of ATRA
(107 to 106 mol/L) can overcome
this block. How might this blockade occur at the molecular level? The
fact that wild-type RAR as well as mutant forms of RAR can cause
the block suggests a squelching mechanism.86 At low ATRA
concentrations, coactivators may bind loosely to nuclear receptor/DNA
complexes and be easily sequestered by high levels of free normal or
aberrant receptor in the nucleoplasm. Only at pharmacological ATRA
concentrations would the cofactors be drawn to target genes along with
RAR and the basal transcriptional machinery. In support of this
notion, in vivo footprinting of the RAR promoter shows occupancy of
the RARE only under pharmacological ATRA concentrations.87
It might be predicted that forced expression of RXR and/or RAR
coactivators would rescue the block by dominant negative RAR and
allow differentiation to proceed at physiological ATRA concentrations.
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RAR TARGET GENES |
ATRA treatment of myeloid precursor cells and other cells drives the
expression of multiple classes of genes
(Table 1) expressed either immediately
after ATRA treatment or after some delay. ATRA-induced changes in
myeloid gene expression are accompanied by inhibition of cell growth
and induction of terminal differentiation and production of a mature
cell ready to fight infection. The retinoic acid syndrome encountered
during treatment of APL with ATRA, characterized by an increase in
leukocyte count, fever, and pulmonary infiltrates, may be due to the
increased adhesive characteristics of the differentiating granulocytes
and secretion of cytokines.88 ATRA also downregulates the
expression of procoagulants found in the undifferentiated promyleocyte,
explaining the ability of differentiation therapy to treat APL without
inducing hemostatic disorders (reviewed in Barbui et al89).
The initial waves of leukocytes found in APL patients are derived from
the malignant clone90 and function normally in vitro to
kill pathogens,91 despite some abnormalities of secondary
granules involved in immune function.92,93
Many of the target genes of RAR that induce rapidly after ATRA are
themselves transcription factors.38 There are RAREs in the
promoters of the RARs (see above) that may help explain the ability of
ATRA to induce differentiation in APL. ATRA treatment of fresh APL
cells upregulates the mRNA for RAR, correlating with the presence of
a RARE in the second promoter of the RAR gene.32,94
Therefore, one way that ATRA may induce myeloid differentiation may be
to upregulate the RAR/RXR complexes, overcoming the dominant negative
PML-RAR protein. This hypothesis is supported by recent data that
indicate that PML-RAR is selectively degraded in APL cells by
treatment with ATRA,95,96 further shifting the balance
towards the wild-type receptor. Other candidates for directly
regulation by the retinoid receptors during myeloid development include
members of the hox family of homeobox-containing transcription factors (reviewed previously97,98). Hox genes are expressed in myeloid cell lines in a coordinated, dynamic manner.99
Enforced expression of the hox genes100-103 or disruption
of their expression104 as in leukemia-associated
translocations105,106 is associated with altered myeloid
growth and differentiation.107 RAREs were identified in the
promoters and enhancers of hoxb1108-110
hoxa1,111,112 and hoxd4.113
Furthermore, specific RARs differentially regulate the homeobox genes.
In embryonic carcinoma cells null for RAR, ATRA fails to induce the
hoxb1,38,114 whereas RAR null cells fail to
express hoxa1. This information suggests that in APL the disruption of RAR function may alter expression of a subset of ATRA-inducible genes critical for myeloid differentiation.
Recent data indicate an interplay between retinoic acid and interferon
(IFN)-mediated signaling. ATRA can rapidly induce transcription of the
IFN regulatory factor-1 (IRF-1) gene. IRF-1 expression is associated
with the expression of IFN and IFN-inducible genes,115 cessation of cellular growth, and induction of apoptosis. Thus, IFN may
potentially mediate some of the antiproliferative effects of
ATRA.116,117 ATRA induction of the IRF-1 promoter is
mediated by a GAS (-IFN activation sequence) rather than an
RARE,118 signifying a role for retinoic acid in the STAT
(signal transducer and activator of transcription) pathway for IFN
signaling.119 ATRA rapidly induces the expression of
STAT1 at the mRNA level and increases tyrosine phosphorylation of
STAT1, together leading to a large increase in DNA binding activity
of the STAT1 complex to an IFN-responsive element
(IRE).120 RAR and STAT1 synergized to stimulate
transcription from an IRE-containing reporter plasmid, whereas
PML-RAR did not, suggesting that cross-talk between the two
signaling pathways may be aberrant in APL and play a role in disease pathogenesis.
Recently C/EBP- , a newly identified basic-zipper transcription
factor that recognizes CCAAT DNA sequences, was found to be rapidly
upregulated during ATRA-mediated differentiation. C/EBP- is the only
member of this family of transcription factors expressed in the APL
cell line NB4,121 suggesting that the gene may play a role
in the promyelocyte stage of differentiation.122-125 HL60 cells engineered to express PML-RAR downregulate C/EBP-
expression in the absence of retinoic acid; the C/EBP- gene is then
upregulated when pharmacological doses of ATRA are added to the
cell.126 Hence, C/EBP- may be a model target gene of the
PML-RAR fusion protein that is inhibited in expression at ambient
physiological concentrations of ATRA and stimulated in expression when
cells are treated with ATRA.
ATRA is known to alter cell cycle kinetics, because it induces the
differentiation of APL and other myeloid cells.127,128 ATRA
treatment is associated with G1 arrest and the accumulation of
hypophosphorylated forms of the retinoblastoma protein.129 ATRA induces the expression of the p21WAF1/Cip1
cyclin-dependent kinase inhibitor in myeloid cell
lines.130,131 ATRA-mediated p21 induction may also depend
on the PML protein disrupted in t(15;17) APL.132 RAR in
combination with RXR binds to an imperfect RARE within the human p21
promoter and RAR can activate the p21 promoter in a ligand-dependent
manner. Therefore, p21 meets criteria for a bona fide RAR target
gene whose expression could decrease leukemic cell proliferation.
Potentially relevant to the treatment of APL, the cytosolic retinoic
acid binding protein II (CRABPII) promoter contains an RARE and can be
transcriptionally induced by ATRA.133,134 CRABPII may
sequester ATRA, contributing to therapy resistance.135
Tissue glutaminase II, which plays a role in differentiation and
apoptosis, is induced rapidly by ATRA136 and contains
functional RAREs in its promoter.137,138
Identification of further direct target genes of RAR relevant to
normal and malignant myelopoiesis has accelerated, using techniques
such as subtractive cloning,139,140 differential
screening,141 differential display (DD),142,143
and representative difference analysis (RDA).144 These
studies focused on genes induced 24 hours after ATRA treatment and
tended to identify indirect targets of ATRA action. These included the
IFN-inducible RIG-G145 and the related RI58
gene,146 RIG-E, encoding a GPI-linked cell surface molecule,147 and Jem1,148 a basic/leucine
zipper transcription factor gene. In contrast, using RDA, a
calcium/calmodulin kinase was isolated from ATRA-treated murine
promyelocyte MPRO cells harboring a dominant negative RAR molecule.
This gene was activated within a few hours after ATRA treatment in a
cycloheximide-resistant fashion and therefore is a reasonable candidate
as a direct target of RAR.149 Another report also using
the MPRO cell line isolated, by subtractive hybridization, several
genes rapidly induced by ATRA treatment.150 One transcript
induced threefold by ATRA treatment was identical to that encoding
LAPTM5, a lysosomal protein expressed preferentially in hematopoietic
cell lines.151 The LAPTM5 promoter contained RAREs, and an
LAPTM5-luciferase reporter gene was inducible by retinoic acid. The
importance of this gene in differentiation is unknown. Current efforts
towards the identification of ATRA target genes are using microarrays
of immobilized cDNAs152,153 or
oligonucleotides,154 which can monitor expression of
thousands of gene simultaneously.155,156
Table 2 summarizes some of the salient
points regarding myeloid biology, RARs, and APL.
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PML |
PML structure.
The t(15;17) rearrangement affecting the PML gene on chromosome 15q22
is the molecular basis for approximately 98% of all cases of
APL.5 The PML gene locus spans 35 kb and contains 9 exons
encoding mRNAs of 4.6, 3.0, and 2.1 kb. Alternative splicing of
C-terminal exons yields up to 20 different isoforms of the protein5; however, most cell lines express a similar
pattern of isoforms.11-13,157-159 The longest cDNA open
reading frame encodes a 560 aa polypeptide with a predicted molecular
weight (MW) of 70 kD.10,159
The PML protein has a modular structure with several domains
(Fig 3). These include the
following:
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| Fig 3.
Functional domains of the PML protein and structure of
the PML-RAR and reciprocal RAR-PML proteins generated in
t(15;17)-APL. In all patients, the RING finger, B boxes, and at least
the first two coiled coil domains of PML are included in the fusion
protein. Heterogeneity in the breakpoint within the PML gene leads to a
long and short form of PML-RAR depicted, as well as the rarer
intermediate form (not shown). The RAR-PML fusion is detected in the
majority of cases, but no evident function can be ascertained from its
structure.
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| Fig 4.
2D-NMR structure of the PML RING finger.166
Spheres representing Zn2+ ions coordinately bind to
histidine and cysteine residues allowing loops of protein to extend in
a spherical configuration (courtesy of K. Borden).
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(1) A cystein-rich region (aa 57-222) composed of three zinc-finger
like structures. The first is a RING (Really Interesting New Gene)
finger,160 a Zn2+ binding motif with the
configuration C3HC4 (aa 57-91). The following two are called B box zinc fingers (aa 140-161 and aa
189-222).12,13,157,159 The RING finger motif161
is found in more than 80 proteins involved in oncogenesis, regulation
of gene expression, mRNA processing, and DNA recombination and repair
(reviewed previously160-162). RING finger/B Box proteins
often are linked to a coiled-coil domain and comprise a subfamily of
RING proteins (RBCC for RING-B Box-Coiled-Coil).
The RING finger/B-box region is involved in localization of PML into
distinct nuclear domains known as nuclear bodies (NB), presumably
through interactions with protein partners (see
below).5,163,164 Biophysical studies, including 2-D NMR,
showed that the RING finger structure is spherically organized around
several loops of protein extending from two Zn2+ ions that
coordinately bind cysteine and histidine residues (Fig 4). This
positively charged structure precludes DNA binding, implying that the
RING domain is required for protein-protein
interactions.165,166 PML with mutations in critical RING
finger cysteine residues loses its characteristic nuclear body
localization and its biological activity as a growth suppressor (see
below),164,166-168 but other charged residues on the
surface of the globular RING structure may also affect NB
formation.169 The B box domains also bind zinc ions and any
mutation of the B-box cysteine residues disrupts NB
formation.164 Neither the RING finger nor the B-box motifs are required for PML to self-associate, suggesting that interactions with other proteins, through the Cys-rich motifs guide PML into the
multi-protein complex of the nuclear body (see
below).163,166,170
(2) A helical coiled-coil region (aa 229-360) consisting of eight
heptad repeats with hydrophobic amino acids at the first and fourth
positions. This region is responsible for multimerization of PML and
heterodimerization with PML-RAR and plays a role in NB localization.
This region also interacts with partner proteins and is required for
the growth suppression activity of PML as well as the ability of
PML-RAR to block differentiation.163,167,170,171 All PML
isoforms contain the RING Finger/B Box domains as well as at least the
N-terminal coiled-coil motifs.
(3) An N-terminal proline-rich sequence (aa 1-46) that can bind the
Arenavirus Z proteins involved in viral genome synthesis172 but is not required for growth suppression by PML.167
(4) A basic sequence, containing a nuclear localization signal (aa
476-490)167,170 required for the biological activity of the
protein.167 However, exact nuclear localization in NBs also requires the RING finger/B box and coiled-coil motifs.
(5) An acidic C-terminal Ser/Pro-rich domain of unknown
function, highly variable in length due to alternative splicing and rich in potential phosphorylation sites.11-13,157,159,167
The PML protein.
PML, when expressed after transfection into cells, is detected as a
series of 90- to 100-kD protein bands as well as a set of bands in the
70 kD range, as predicted from the amino acid sequence.173-175 Endogenous PML is detected as a 90-kD
species along with a variety of other protein species (150-50 kD) due
to alternative splicing and covalent
modifications.174,176-180 The PML sequence contains
potential casein kinase II and proline-directed kinase sites, and
32P-labeling studies demonstrated that PML is
phosphorylated on serine and to a lesser extent on tyrosine
residues.5,170,174,181 Some of the sites are constitutively
phosphorylated and others may be cell cycle dependent.182
In this regard, PML was found to be a substrate for phosphorylation by
Cyclin A/cdk2174,182 in vitro.
PML expression.
PML mRNA was widely expressed in all cell lines
tested.5,183 The pattern of PML protein expression in
tissues is complex and controversial, possibly due to differences in
techniques and antibodies used for immunodetection. In addition, PML
mRNA and protein expression are often not concordant, suggesting
posttranscriptional regulation. There are a few observations that
should be highlighted.
First, PML is highly expressed in inflammatory diseases such as
psoriasis and hepatitis, in inflammatory cells surrounding epithelial
cancers and Hodgkin's disease, in inflammatory lesions of
graft-versus-host disease, and in activated
fibroblasts.183-186 This suggests induction of PML
expression by soluble factors, probably IFNs (see below). Second, some,
but not all,185 groups found a correlation between the
level of PML expression and degree of dysplasia in atypical breast
hyperplasia and cervical intraepithelial neoplasia cells.
Interestingly, when the breast tumors became invasive, PML expression
decreased again.183,184 Some investigators found a
correlation between the rate of proliferation of normal tissues and PML
expression, with postmitotic cells tending to express higher levels of
PML,175,184,185 whereas others187 suggested that PML levels were better correlated with cellular activation by
cytokines and rates of protein synthesis. Third, PML delocalization can
be associated with neoplasia. Hepatic carcinoma in particular was
associated with mislocalization of PML in the cytoplasm rather than the
nucleus.183 Fourth, PML and probably other proteins of the
nuclear body are induced by hormones such as estrogen and cytokines
such as IFN.177,183,184,188,189 Lastly, PML is expressed in
myeloid precursors in the bone marrow173,189 and to a
lesser extent in circulating monocytes and granulocytes. Activation
with IFN leads to reinduction of PML in these cells. This suggests an
early role for the protein in myeloid differentiation, perhaps regulating cell growth, and later in host
defense.173,175,183,185,190 In lymphoid cells, PML is
expressed mainly in postmitotic mature T and B cells and not the
germinal center or in proliferating cortical thymocytes.175
Hence, PML may play a broad role in mature immune cells.
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NUCLEAR BODIES AND PML EXPRESSION |
One of the most striking features of PML is its speckled localization
to discrete nuclear domains termed PODs (PML Oncogenic Domains), ND10
(Nuclear domain-10), or NB.170,173,191,192 These structures, originally described more than 35 years ago,193
came under new scrutiny when NB proteins, including PML, were detected by human autoimmune antisera.191,194-199 Nuclear bodies
vary in both size and number in different cell types. Their presence is
roughly proportional to the rate of protein synthesis and inversely
proportional to differentiation.187 Strikingly, PML is
delocalized from the NBs to a microspeckled nuclear pattern in t(15;17)
APL cells and relocalizes to the NB after ATRA
treatment.173,191,198 Cells generally contain 10 to 20 doughnut-like or spherical 0.3-0.5-µm NB structures. Electron
microscopy showed that, in the NB, PML and other proteins surround an
electron dense core that may contain ribonucleic
acid.197,198,200 NBs are associated with the nuclear matrix, which plays a role in trafficking of molecules and organization of chromatin within the nucleus. In initial studies, the NBs did not
overlap with spliceosomes, centromeres, or sites of RNA
transcription.191,197,201 Neither sequence-specific
transcription factors such as the glucocorticoid receptor and E2F nor a
general factor such as TFIIH were concentrated within the
NB,201 which appeared to exclude a transcriptional role for
the NB. This notion must be seriously re-examined in light of new data
from the lab of Ron Evans, who, using refined techniques, found the
presence of nascent mRNA in the center of the NB
structure.202 Furthermore, the transcriptional coactivator CBP was found colocalized in the NB with PML. This, together with recent data indicating the presence of PML in the AP1 DNA binding complex,203 suggests that the NB may play a role in
stimulating transcription. Whether the NB might be a site of
transcriptional initiation, elongation, or processing of the new mRNA
transcript is unknown. However, data that PML can repress transcription
and the finding of HP1, a heterochromatin component, within the
NB204 suggest a role for PML and/or other NB components in
downregulation of transcription.
PML-containing nuclear bodies do not colocalize with sites of nascent
DNA,191,197 except during mid-S phase, when they are found
adjacent to replication sites,201 potentially indicating a
role for the NB in this process. Although PML-containing nuclear bodies
are distinct from the coiled body, involved in mRNA
splicing,205 the two structures were often found adjacent
to each other, suggesting a functional
interrelationship.201 PML was also found in the interchromatin granules, which are thought to be sites of RNA splicing,206 and can be found in the nucleoplasm in a
diffuse staining pattern192 as well as in a cytoplasmic
granular pattern.175,189 Although the exact site and
mechanism of PML action is still uncertain, nuclear expression of PML
seems to be essential for its biological function, because PML mutants
lacking the NLS were unable to suppress oncogenic
transformation.167 However, certain spliced isoforms of
PML, devoid of the C-terminal NLS, are found exclusively in the
cytoplasm,175 but other isoforms that do contain the NLS can be found in both the nucleus and cytoplasm of transfected cells.
Hence, a cytoplasmic role for the protein is not ruled out and neither
is the possibility that PML may shuttle between the nucleus and
cytoplasm. The partition of PML between the nucleoplasm and the NBs may
be controlled by processes such as differential phosphorylation and
conjugation of PML to a ubiquitin-like molecule, sentrin.179
The presence of the RING finger, B box motifs, and coiled-coil motifs
are all necessary for PML to properly localize in the NBs.167 Mutants lacking these structures sequestered normal
PML from the NBs in a dominant negative manner.167,175 In
addition, forms of PML with mutations in critical cysteine residues
required for normal protein folding failed to localize in the
NBs.164,166,170 PML expression and NB structure are
dynamic. Augmenting the cellular levels of PML either by transfection
with a PML expression vector or by treating cells with IFNs increased
the size and number of nuclear bodies, possibly due to deposition of
PML and recruitment of other proteins to these
sites.177,189,192,198,207 In contrast, PML-RAR caused
disappearance of nuclear bodies from the nucleus as determined by
staining with antibodies directed against PML and the SP100 NB
protein.198
The pattern and intensity of PML expression changes across the cell
cycle. Cells in G0 exhibit few nuclear bodies and show weak
staining for PML. As cells are stimulated into the cell cycle, the
number of NBs and their intensity of staining with PML antibodies increases. As cells progress through S to G2 phase, PML disperses to
multiple smaller dots and gradually fades, with two or three of these
residual structures left in mitosis.174,183,184 Prolonged amino acid starvation208 or induction of cell
senescence209 induces the coalescence of the NBs into 2 to
3 large structures. When amino acid-starved cells were rescued by
addition of fresh nutrients, the normal pattern of 10 to 20 smaller NBs
reappeared.208 The pattern of PML expression also responds
to cell stresses such as heat shock,192
-irradiation,200 and viral infection (see below). How
these changes relate to the function of the nuclear body is unknown.
| |
PML AND TRANSCRIPTION |
Findings related to transcriptional properties of PML have been
contradictory but intriguing. PML specifically suppressed transcription
of the MDR and EGF-receptor (EGF-R) promoters.210 However,
PML enhanced transcription of the CD18 promoter210 as well
as the promoter for the gene encoding the major histocompatibility complex (MHC) class I transporter molecule
TAP-1.211 Furthermore, PML increased the transcriptional
activity of the progesterone receptor (PR) in the presence of hormone
up to 20-fold and also stimulated transcription mediated by the
mineralocorticoid, glucocorticoid (GR), and androgen receptors, but not
the RARs.212 Deletion analyses indicated that both
activation domains (AF1 and AF2) of PR as well as the Cys-rich and
coiled-coil domains of PML were required for the PML effect on
transactivation. PML did not increase the affinity of PR for
progesterone, increase DNA binding by PR, or coprecipitate with PR.
This does not preclude PML as a transcriptional cofactor or adaptor to
the basal transcriptional machinery. Alternatively, PML might sequester
a negative regulatory factor from the PR, indirectly stimulating its
activity. The physiological relevance of this interaction must be
tempered by a report that PML does not colocalize with the
GR.201
GAL4-PML fusions were found to repress a reporter gene containing GAL4
binding sites,213,214 although an earlier publication did
not note such an effect.170 The repression domain mapped to
the coiled-coil region and the C-terminal serine-rich region but not to
the RING finger; the magnitude of repression was cell-type and promoter
specific. Mutational analysis indicated that the coiled-coil, not the
RING finger motif was required for repression and that localization to
the NB was not required. Rather, the nucleoplasmic fraction of PML may
be responsible for this effect. It is tempting to speculate that PML,
like PLZF (see below), interacts with histone deacetylase corepressors
to repress transcription. Another mechanism of action of PML was
suggested by the finding that PML-mediated repression of the EGF-R
promoter was mediated through Sp1 sites. PML was then found to inhibit
the DNA binding by Sp1 protein.215 These data suggest that,
under certain circumstances and probably not when localized to the
nuclear body, the PML protein could modulate gene expression by direct
interaction with specific transcription factors. This function might be
abrogated by PML-RAR, which, when coexpressed with Gal4-PML, leads
to simulation of transcription rather than repression.214
Although PML might be a transcriptional corepressor, a recent report
also indicated that PML can stimulate transcription mediated by fos and
jun through AP1 sites.203
| |
PML PARTNER PROTEINS |
The matrix association of PML makes it difficult to study the
interactions of PML with putative partners, because aggressive purification procedures required to extract PML from the nuclear matrix
can disrupt protein-protein interactions.216 It is
interesting to note that two other RAR translocation partners, NPM
and, in particular, NuMA, are also closely associated with the nuclear matrix. In addition, the remaining fusion partner, PLZF, is also found
in (the matrix-associated) nuclear bodies. Together, these observations
indicate that disruption of nuclear matrix function may be a common
theme in APL pathogenesis, perhaps further disturbing normal patterns
of gene expression.
Nearly 20 natural components of the nuclear body have been identified
to date (Table 3). The diverse nature of
these proteins and the fact that they often localize both within the
nuclear body and other subnuclear patterns suggests that the NBs may
not be a site of active cellular metabolism but rather a storage site for nuclear proteins whose temporal and spatial expression must be
tightly controlled. However, several of these proteins are induced by
IFN, including PML, SP100, and PLZF,216-219 pointing to an
alternative hypothesis that the nuclear bodies may play a role in
cellular proliferation and/or the antiviral response.
Only a few proteins are known to actually bind directly to PML. The
first of these, sentrin, originally called PIC1 (also called SUMO-1 and
UBL-1), was identified in a yeast two hybrid screen.220
Both cotransfected and endogenous PML and sentrin colocalized in the NB
with variable overlap. In the APL NB4 cell line, sentrin was partially
delocalized to PML-RAR microspeckles. Sentrin is a ubiquitously
expressed 11.5-kD peptide containing a ubiquitin homology (UbH)
domain.220 Two other groups independently cloned sentrin by
its association with the rad51 DNA repair protein221 and
the ranGAP protein of the nuclear pore complex,222 whereas a third group found that sentrin interacted the tumor necrosis factor
(TNF) receptor. This group found that sentrin covalently attaches to
proteins in a manner akin to ubiquitin, suggesting that sentrin may
play a role in protein turnover.223 Furthermore, this group
found that overexpression of sentrin protected cells from fas/apo- and
TNF-induced apoptosis.224 Given the role of PML as a growth
suppressor (see below), it is possible that PML sequesters sentrin,
promoting apoptosis and balancing the function of sentrin. However, it
is more likely that sentrin modifies the function of PML and other
components of the nucleus. Newer data show that sentrin covalently
binds to PML in the RING finger motif, the B box domain, and the more
C-terminal nuclear localization signal of
PML.178,180,225,226 This association was exclusively found
in nuclear localized PML, particularly with PML associated with the
NBs,225,226 suggesting that the sentrin modification targets PML to the NB. However, sentrin was not associated with PML in
mitotic cells, suggesting that the modification is cell cycle
dependent. It was reported that PML-RAR is not modified by
sentrin180,226 and that sentrinization was not restored to this protein after ATRA treatment. This was surprising given the moieties modified by sentrin are present in PML-RAR, and another group found that, when expressed in U937, PML-RAR but not
PLZF-RAR became conjugated to sentrin, suggesting that the molecule
might play a role in the degradation of PML-RAR after ATRA
treatment.227
The PLZF protein may be a key interaction partner of PML. The PLZF
protein, originally characterized by its fusion to RAR in
t(11;17)(q23;q21)-associated APL, colocalizes with PML both in myeloid
and in transfected nonhematopoietic cells.216
Colocalization was incomplete, and one group estimated only
approximately 30% overlap of PML and PLZF in KG1 cells.228
The PLZF nuclear dots and PML NBs also appeared to be in different
functional compartments, because expression of the adenovirus E4
protein in KG1 cells delocalized PML from the NB (see below) but did
not affect the expression pattern of PLZF. In semistable transfected
cells, several days were required for the PML and PLZF proteins to sort
within the cell and become colocalized, suggesting complex regulation
of this association.216 Nevertheless, interaction between
PML and PLZF was demonstrated by biochemical assay, and the association was found to be mediated by the coiled-coil domain of PML and the first
two zinc fingers of PLZF (A. Zelent, personal communication, December
1998). PLZF could be delocalized to a microspeckled pattern in NB4
cells and reverted to the NBs upon treatment with retinoic acid. These
provocative findings suggest that PML may be involved in the
transcriptional modulation mediated by PLZF or, conversely, that PML
regulates availability of PLZF by sequestration within the
NBs.216 Thus, a common mode of leukemogenesis may exist in APL, based on disruption of a pathway that contains both PLZF and PML.
New data indicate that the retinoblastoma (Rb) protein interacts with
PML. PML could be coimmunoprecipitated with a low percentage of the
underphosphorylated form of Rb but not the related p107 and p130
proteins. As seen by confocal microscopy, the speckled fraction of Rb
in the cell overlapped the PML NB. PML-RB interaction required the
pocket domain of Rb and the first two coiled-coil motifs of PML, with
the RING finger motif, stabilizing the complex. In addition, a PML
isoform with an extended serine-rich C-terminal domain bound
significantly less strongly to Rb, suggesting that different PML
isoforms could differentially bind Rb.229 PML-RAR also
bound to Rb and delocalized Rb from its usual speckled pattern into the
microspeckled pattern characteristic of PML in APL. The functional
consequences of this interaction in growth control are uncertain,
because both PML and PML-RAR are able to inhibit the growth of
nonhematopoietic cells devoid of functional Rb. However, it remains
fascinating to postulate that one of the mechanisms through which PML
inhibits cell growth is by limiting accessibility of Rb to the
cyclin-dependent kinases that phosphorylate it during G1 phase.
Finally, a novel function for PML was suggested by its association with
the ribosomal P proteins P0, P1, and P2.230 These proteins
form part of the large ribosomal subunit, are localized in both the
nucleus and cytoplasm, and are involved in the process of
translation,231-233 suggesting that PML may have a role in
translational control. This is supported by the findings that PML
interacts with the L7 leucine zipper protein and EF-1,220
also implicated in translation, and that PML partially localizes to the
cytoplasm.175 It is intriguing to note that the NPM
protein, fused to RAR in t(5;17) APL,234 is involved in
ribosome biogenesis and shuttling ribonucleoproteins between the
nucleus and cytoplasm.235-237 This places both PML and NPM
in a similar functional axis.
| |
PML, IFN, AND VIRAL INFECTION |
PML is an IFN-responsive gene that may be important for its normal
immunity. In hematopoietic NB4 and nonhematopoietic cell lines, PML and
PML-RAR mRNA were induced fivefold to 30-fold in response to IFN in
a dose-dependent manner.177,188,189 Actinomycin D blocked
this effect, whereas cycloheximide did not, indicating that PML is a
primary target of IFN action. Other cytokines that induce the JAK/STAT
pathway did not cause this increase. The PML promoter contains an ISRE
(IFN-stimulated response element) as well as a GAS ( activation
site), both of which bind to STAT proteins.158 After IFN
treatment, different PML isoforms become visible with increases in the
intensity of staining and number of nuclear bodies. STATs are involved
in upregulating multiple components of the NB, including Sp100, whose
promoter also contains a GAS and ISRE.176 Because PML seems
to be a growth suppressor, it may be an attractive target for the
antiproliferative and antiviral effects of IFN.158 Finally,
ATRA can cooperate with IFN to synergistically induce PML expression
through induction and activation of STATs acting through the GAS
element in the PML promoter.118 Induction of wild-type PML
could thus play a role in the therapeutic effect of ATRA in APL.
It is apparent that one of the earliest events of both lytic and latent
viral infections is the targeting of viral products to NBs, often
resulting in the reorganization of PML localization and NB
architecture. Some of these results are summarized.
Adenovirus.
Adenovirus infection causes PML to undergo dynamic spatial
redistribution. As the virus progresses through its life cycle, the NBs
disappear and become part of the viral nuclear
inclusions.206 Mutational analysis showed that the
adenovirus E4-ORF3 gene product was responsible for NB redistribution
to fibrous structures or tracks. Overexpression of PML blocked this
redistribution, an effect that was lost when PML deleted for the
coiled-coil domain was used.238
HSV-1.
ICP0, an immediate early gene, encodes a RING finger protein that is
required for both the lytic and latent herpes virus life cycles. ICP0
activates viral gene expression, targets the NBs, colocalizes with PML,
and modifies NB architecture.239 HSV-1 replication sites,
with attachment of viral DNA to the nuclear matrix, appear to coincide
with NBs as well,240 suggesting that NB components may be
involved in viral DNA replication. In latent infections, in which HSV
genes are partially expressed from episomes, viral genomes may also be
associated with NBs. Stress, which disperses the NBs, also allows the
virus to come out of latency.240
Epstein-Barr virus (EBV).
In contrast to the viruses listed above, EBV tends to transform
lymphocytes by latent infection rather than lysing target cells. EBNA 5 is an early protein critical for transformation and latency. This
protein is colocalized with PML in interphase NBs, with PML coating the
outside of the structure and EBNA found within.241,242 In
contrast to the other viral infections, the NB structure is not
disrupted by EBV infection.
Arenavirus (LCMV, Lhassa).
The arenavirus RING finger containing Z protein binds to the N-terminal
proline-rich region of PML, resulting in its delocalization to the
cytoplasm.172 The fact that the Z protein targets P
ribosomal proteins that are partners of PML suggests viral hijacking of the cellular translational machinery, of which PML may be a
component.230 Alternatively, PML may be moved to the
cytoplasm to prevent it from promoting apoptosis (see below), thus
enabling survival of virus-laden cells and allowing the virus to
establish chronic infections.168,230 This list is by no
means complete, because cytomegalovirus
(CMV)243,244 and papilloma
virus245 also target the NBs.
To test whether PML plays a role in the antiviral effect of type I IFN,
PML / embryonic fibroblasts were infected with a number of
DNA viruses, including herpes simplex and vesicular stomatitis virus,
in the presence or absence of IFN.207 There was no
difference in the efficacy of IFN in decreasing resultant viral titers;
therefore, the absence of PML did not confer increased susceptibility
to viruses. Engineered expression of PML in 3T3 cells did not confer
resistance to HSV, VSV, EMCV, or Adenovirus.207 However,
another group found that in HepG2 cells, overexpression of
PML inhibited adenovirus growth, perhaps by delaying or blocking NB
protein recruitment to viral replication domains.238 A
third group found that PML overexpression selectively inhibited
replication of influenza virus but not of encephalomyelitis virus. In
contrast, overexpression of the Sp100 NB protein had no significant
antiviral effect.246 Together, it can be concluded that,
although PML itself is not absolutely required for the activity of IFN,
it may confer some of the antiviral activity of IFN.
These findings suggest that NBs could be carefully controlled
organelles that store regulatory factors involved in viral
transcription or replication.239 Alternatively, NBs may be
an intranuclear defense system. This notion is supported by the
localization of infecting virus genomes to the vicinity of the NB, the
IFN induction of NB proteins, and the growth-suppressive properties of
PML.240 Perhaps more plausibly, viruses may target and
disrupt the nuclear body to abrogate an apoptotic program (see below),
allowing the virus to promulgate freely in the cell population. The
delocalization of NB proteins caused by PML-RAR expression could
mimic activation of the viral program, possibly resulting in
uncontrolled cellular proliferation.238
| |
PML, GROWTH SUPPRESSION, AND APOPTOSIS |
The growth-suppressive actions of PML were suggested by the finding
that NB4 cells selected for expression of exogenous PML harbored
mutations in PML, suggesting that the wild-type protein was not
tolerated.210,247 Infection of NB4 cells with a retrovirus harboring PML suppressed the ability of the cells to form colonies in
soft agar. In addition, conditioned medium from these cells suppressed
colony formation of wild-type NB4 cells, suggesting the release of
negative growth control factors.210 Furthermore, PML-overexpressing NB4 cells, when injected into nude mice, yielded smaller tumors that appeared with a longer latency than
vector-expressing cells.210 Attempts to circumvent the
toxic effect of PML led another group to create an episomal
transfection system in which it was demonstrated that even low levels
of PML were toxic to NB4 cells. Because RAR overexpression was also
growth suppressive to these cells, these investigators concluded that,
in t(15;17) APL, the reduced dosage of PML and RAR could contribute
to uninhibited cell growth.247
It was further shown that PML inhibited transformation of rat embryo
fibroblasts expressing Ha-ras and mutant p53 or Ha-ras and
c-myc.210 However, PML did not inhibit all oncogenic
pathways. PML suppressed foci formation in NIH 3T3 cells transformed by the neu oncogene, but not by the ras oncogene. PML-RAR acted in a
dominant negative manner to prevent suppression of transformation by
PML, sequestering PML in the cytoplasm, whereas PML-RAR itself did
not stimulate the formation of foci. The neu oncogene-transformed NIH-3T3 cells mentioned above were reverted by retrovirus-mediated transfer of PML, resulting in restitution of wild-type morphology, contact inhibition, and anchorage-dependent growth. There was no major
difference in cell cycle distribution of PML-expressing cells,
suggesting that PML suppressed growth not by cell cycle inhibition but
by altering cell survival and apoptosis. Although PML expression led to
decreased expression of the neu protein, the cell cycle profile and
morphologic response of cells to expression of PML differed from that
of cells treated with an anti-neu antibody, which arrest in the G2
phase of the cell cycle. This suggests that PML interferes with
downstream targets of the neu signal transduction molecule, perhaps
reflective of the normal physiologic function of PML.248
However, in another system, PML did affect the progression of the cell
cycle. When overexpressed in HeLa cells, PML inhibited cell growth,
colony formation in agar, and tumor growth in nude mice, as in the
models described above, but also caused an accumulation of cells in
G1249 and a delay of cell entry into S phase, correlated with decreased expression of cyclin E and cdk2. Similarly, expression of PML in breast cancer cell lines blocked cells in G1
associated with a decrease in cyclin D1 and CDK2 and accumulation of
hypophosphorylated Rb.250 This is intriguing given recent
data that hypophosphorylated Rb and PML can complex.229
However, PML can suppress growth even of Rb-defecient cells, suggesting
that complexes with other Rb-like proteins could play a role as well.
de Thé's group stably expressed PML in HeLa, CHO, and NIH 3T3
cells and observed a twofold to fivefold decrease in growth rate,
decreased colony formation, and inhibition of tumor formation in nude
mice.184 Growth suppression by PML was further augmented by
IFN, perhaps by stimulating expression of other nuclear body proteins.184 A structure/function analysis of the PML
protein indicated that deletion or mutation of the RING finger motif of PML abrogates both NB formation and growth
suppression.166-168,170 Deletion of the coiled-coil motif
yielded a diffuse nuclear pattern of expression and no growth
suppression, whereas deletion of the NLS of PML or expression of a
splice variant of PML leads to a granular cytoplasmic pattern of
expression and also abolished growth suppression.251 The
proline-rich region at the N-terminus of PML and the serine proline
variable region at the C-terminus of the protein were dispensable both
for NB formation and growth suppression.167 Thus, PML
expression in the nucleus is required for growth suppression, although
a recent report questions whether expression in the NB per se was
required for growth suppression.251 In support of this
observation, infection of NIH 3T3 cells with LCMV virus rapidly
relocalized PML into the cytoplasm and delayed apoptotsis after serum
starvation.168 Similarly, treatment of cells with antisense
PML oligonucleotides delayed death after serum starvation. These
results suggest that the correct localization of PML plays an important
role in apoptosis and cell growth.
Recent data suggest that PML may promote cell death by novel
mechanisms. PML interaction with the P0 ribosomal
protein230 could target the 28S rRNA for cleavage, an
important step during apoptosis.230 PML expression yields
cell death that is not associated with the usual chromatin condensation
or activation of caspase 3 the major effector of
apoptosis.252 Paradoxically, zVAD a caspase inhibitor
accentuated PML-mediated apoptosis and increased PML expression levels
in the NB, as did arsenic (see below). PML was found to recruit the
pro-apoptotic BAX protein to the NB as well as the cell cycle inhibitor
p27KIP1; the importance of this recruitment remains
unclear, because it is still not certain what processes occur in the
NB. Use of PML/ animals yielded somewhat different
results, with a 50% decrease in apoptosis of T cells after
-irradition and a great reduction in fas-induced
apoptosis.253 PML null mice had no significant difference
in expression of multiple apoptosis mediators, with the exception of
caspase 1 and 3, whose activities, required for radiation and
fas-mediated apoptosis, were markedly reduced. Furthermore, PML null
animals were resistant to ceramide, TNF, and IFN-mediated apoptosis.
Together, these data indicate a critical role for PML in multiple
apopotic pathways. High-level expression of PML may induce apoptosis in
the absence of caspase action, whereas physiological levels may be
required for the normal activation of caspases.254
It is certain that PML is growth suppressive when overexpressed, but
whether it is a tumor suppressor is uncertain. Mutations of PML have
not been reported in other forms of cancer; even in t(15;17) APL,
patients would not be expected to be null for this protein. However,
new data from experiments with targeted disruption of the PML gene
support a tumor-suppressor function for the protein.132,190 PML/ mice were viable and fertile but highly susceptible
to fungal or bacterial infections despite a strong inflammatory
response,132 suggesting a functional defect in the
inflammatory cells. Circulating and bone marrow mature myeloid cell
counts were modestly decreased in these animals, indicating that,
although PML was not required for the formation of the myeloid lineage,
it may be involved in efficient terminal differentiation of these
cells. Early death of these mice from infection prevented the long-term
follow-up required to detect spontaneous tumor formation. However, the
skin of PML/ animals treated with the chemical tumor
initiator DMBA formed twice as many papillomas as that of wild-type
animals. In addition, animals treated with DMBA systemically formed
twice the number of lymphomas as PML replete animals. PML null murine embryo fibroblasts proliferated more rapidly than wild-type fibroblasts and more readily formed colonies in soft agar. Whereas ATRA suppressed the growth of wild-type fibroblasts, there was little effect on the
PML/ cells. Whereas IFN inhibited the proliferation of
normal marrow precursors as determined by clonogenic assays, -IFN or -IFN had no myelosuppressive effect on PML/
marrow.132
PML may inhibit tumor growth in other ways as well. In a remarkable new
study, PML was found to stimulate expression of MHC class I antigens
and the transporters responsible for moving peptides to the cell
surface in association with class I antigens.211 Hence,
disruption of PML expression, whether by tumor viruses such as
adenovirus or by the PML-RAR fusion protein, might lead to decreased
presentation of viral or oncoprotein antigens and defective immune
surveillance for tumors. A recent study implicates PML in growth
control by p53. PML expression was induced fivefold to 10-fold at the
posttranscriptional level by ionizing radiation255 or a
DNA-damaging agent. Overexpression of p53 in HeLa cells was also
correlated with PML induction and G1 arrest, suggesting that PML might
be considered a GADD (growth arrest and DNA damage) gene.256
These exciting data suggest that PML can inhibit cell growth and may be
required to mediate some of the physiological actions of the IFNs. To
study potential effects of PML on RAR signaling, an important target
gene of RAR in differentiation, p21WAF1/CIP1,
encoding a cyclin-dependent kinase inhibitor, was studied.
Interestingly, p21 WAF1/CIP could not be upregulated in the
PML/ fibroblasts. Thus, PML might be required for certain
pathways of retinoid signaling. Therefore, PML-RAR might also
disrupt RAR function in APL by blocking the ability of wild-type PML
to cooperate with RAR to stimulate myeloid differentiation. The L7
leucine zipper protein, a PML partner,220 modulates the
transcriptional activity of nuclear receptor signaling
complexes.257,258 It is therefore conceivable that this
might mediate the effects of PML on retained nuclear receptor function.
In view of the emerging evidence, it is reasonable to describe PML as a
tumor-suppressor protein involved in the growth suppression, differentiation, and immune response pathways of certain cytokines such
as IFN. The mechanism by which PML encourages growth arrest is unclear,
although interaction with Rb and related proteins offers an intriguing
avenue. PML induces apoptosis in the absence of new protein
synthesis252 and may act in both a caspase-dependent and
-independent manner,254 suggesting that its role in
transcription may be secondary to its role in growth control. Apoptosis
by PML is intimately related to its localization in the NB organelle. The NB may be a repository for growth and apoptosis regulators, released or sequestered according to environmental cues, and could be a
key component of the cell's defense system against viral infections.
Delocalization of PML by PML-RAR may be a critical step in the
pathogenesis of APL. Supporting this notion, PML-RAR inibited
fas-mediated suppression of myeloid growth. When expressed in the
PML+/ background, PML-RAR further inhibited apoptosis, suggesting that PML-RAR works in part by subverting normal PML function.253 In opposition to this view, the other forms of
APL occur without removal of PML from the NB, and differentiation block
by PML-RAR can occur without delocalization of PML. If the
PML-RAR fusion is a novel gain of function mutant and/or makes a
critical interaction with another NB protein also in an apoptotic
pathway, then the presence or absence of PML becomes irrelevant to
leukemogenesis. A balanced view suggests that PML-RAR itself is
leukemogenic, but the tumor-suppressor function of wild-type PML acts
to oppose this effect. This is supported by recent experiments crossing
PML-RAR transgenic and PML null mice. APL could indeed develop very
rapidly in the PML null background, more slowly in PML/+ mice,
and with the longest latency in PML+/+ mice.259
| |
PML-RAR |
Structure.
Translocation (15;17) fuses the RAR and PML genes and generates a
PML-RAR fusion transcript (Fig 3). Cases of APL that have morphologically normal chromosomes or other cytogenetic
abnormalities usually have cryptic rearrangements of PML and
RAR.12,260-264 PML-RAR cDNA was cloned from libraries
derived from leukemic blasts of APL
patients.11,13,15,157,170 Comparison of the cDNA structures obtained by multiple groups showed variation in the amount of PML
sequences included in the fusion protein. The RAR portion was
invariant, containing the DNA-binding and ligand-binding motifs (B-F
domains).265 The PML sequence variation seen among patients was generated by heterogeneous breakpoint cluster regions as well as by
alternative splicing.265-269 The most 5' breakpoint,
bcr3, fuses PML exons 1 through 3 of PML, encoding the RING,
B-boxes, and coiled/coil domains to RAR exon 3, encoding the B
domain of the receptor. This breakpoint yields short PML-RAR fusion proteins [PML(S)-RAR]. Bcr1 is more 3' within the
PML gene and includes sequences from PML exons 5 and 6. This fuses up
to 554 amino acids of PML to RAR and has C terminal serine-rich
sequences of PML that are putative sites of phosphorylation
[PML(L)-RAR]. Breakage in bcr2 involves sites in and
around exon 6 of PML and leads to an intermediate length of PML
sequence [PML(V)-RAR]. In general, 70% of patients exhibit
PML(L)-RAR, 20% PML(S)-RAR, and 10%
PML(V)-RAR,270,271 with PML(S)-RAR and PML(L)-RAR representing extremes of contiguous PML sequence fused to RAR. Internal splicing of portions of PML exon 3 led in one patient to a
small PML-RAR fusion protein that contained the RING fingers, B
boxes, and the first two portions of the -helical coiled-coil domain, representing the minimal PML moiety required for
oncogenicity.265 Each t(15;17) APL patient exhibits a
unique set of PML-RAR fusion products indicative of a single
breakpoint with alternative splicing, highlighting the clonal nature of
the disease. Detection of the PML-RAR fusion transcript by reverse
transcription-polymerase chain reaction (RT-PCR) is a
sensitive272 and specific test for the diagnosis of APL and
can be used to measure minimal residual disease after chemotherapy,
differentiation therapy, and bone marrow
transplantation.273,274 Reappearance of PML-RAR
transcripts in the marrow often precedes a frank leukemic
relapse.5,275-277 Initial studies indicated that patients
treated with ATRA who harbored the PML(S)-RAR had a high likelihood
of early death or relapse.278,279 One in vitro study
indicated that blasts from APL patients with the PML(V)-RAR isoform
had decreased ATRA sensitivity.280 There was an association
between the PML(S)-RAR isoform and more primative
morphology281 and secondary cytogenetic abnormalities, suggesting a biological difference between the isoforms,271
possibly due to abnormalities of DNA repair or cell cycle control.
Despite the potential differences among the PML-RAR isoforms,
numerous studies reported consistently good clinical outcomes in all
APL patients,271,282,283 probably due to the highly
effective nature of current therapy.
The mechanism by which the t(15;17) translocation occurs is not known.
It may be that many illegitimate recombinations occur during normal
cell division and are eliminated by DNA repair
systems.284,285 Recent analysis found short stretches of
identity between the PML and RAR genes in the breakpoint
regions.286 It was proposed that random cleavage of the
RAR and PML genes is followed by limited pairing of short stretches
of homologous DNA, repair of the breaks, and joining of the loci. Those
clones that contain the PML-RAR transcript survive and have a growth advantage.
The PML(L)-RAR fusion transcript yields proteins of 110 and 120 kD
and PML(S)-RAR species of 103 and 90 kD,170,174,287 possibly resulting from alternative start
codons.11,13,15,157,170 The fusion proteins of transfected
cells or NB4 cells are about 10 kD larger than proteins generated by in
vitro translation, suggesting the presence of posttranslational
modifications.170,287 In APL cells, PML-RAR is present
in great excess over wild-type RAR, making it the predominant
retinoid receptor in those cells.265,287
Protein-protein interactions.
PML-RAR oncoprotein, an aberrant retinoid receptor with altered DNA
binding activity,11,13,15,157,170,171 can bind RAREs as a
homodimer,171,287 whereas wild-type RAR
cannot.288 Homodimerization requires the coiled-coil domain
of PML and not the E/F ligand binding/dimerization moiety of RAR
(Fig 3). The first 2 (of the 4) hydrophobic clusters of the coiled-coil
region also mediate PML-RAR/PML association. The smallest PML-RAR
protein identified contained only clusters 1 and 2 (Fig
3).265 PML-RAR homodimers can be detected as a distinct
DNA-binding species in nuclear extracts from NB4 cells287
and display weaker affinity for certain artificial and natural RARE
sites than RAR/RXR heterodimers.287 When combined with RXR,
PML-RAR forms multimeric complexes on the
RARE,171,287,289 and even a 1:1 molar ratio of in vitro
translated RXR to PML-RAR favors the formation of PML-RAR/RXR
heterodimers.171,289 Hence, the existence of the PML-RAR
homodimer complex in NB4 extracts probably reflects the high level of
expression of PML-RAR relative to wild-type RAR and RXR in APL
cells. When bound to RAREs along with RXR, PML-RAR displays the same
binding site preference as wild-type RAR. The PML-RAR/RXR
interaction does not require the DNA binding domain of the RAR
moiety within PML-RAR,171 but can occur through the E/F
domain of RAR. Multimeric complexes from transfected cells may
reflect the ability of PML-RAR/RXR heteromers on one DNA binding
site to associate through the PML coiled-coil domain with heteromers on
other sites. In the cell, this could reflect the ability of PML-RAR
to efficiently bind to RXR and sequester it from wild-type RAR.
These multimeric PML-RAR/RXR complexes were also seen on EMSA in
extracts derived from NB4 cells.287 In addition,
PML-RAR, detected by size exclusion chromatography by its ability to
bind [3H]-ATRA, elutes with an apparent MW of 600 to
1,300 kD, further supporting the idea that PML-RAR multimerizes with
itself and/or other proteins.290,291 Reinforcing these
studies, confocal microscopy showed that PML-RAR draws RXR from its
usual subnuclear localization into the compartment occupied by
PML-RAR.170
Taken together, these data suggest that PML-RAR may affect
ATRA-mediated signaling through several mechanisms: (1) binding of
PML-RAR homodimers to a novel set of target genes, (2) binding of
PML-RAR as homodimer or heterodimer with RXR to RAR target genes
in competition with RAR, and (3) high levels of PML-RAR in APL
cells could sequester RXR and/or other RAR cofactors in a novel
nuclear and/or cytoplasmic compartment.
| |
TRANSCRIPTIONAL ACTIVITY OF THE PML-RAR FUSION PROTEIN |
The PML-RAR protein has altered transcriptional properties. Many
groups observed that, in the absence of ATRA, PML-RAR represses transcription from RAREs to a greater extent than
RAR.13,170,292 This may be the most important quality of
PML-RAR. There were conflicting reports regarding transcriptional
activation by PML-RAR. In some reports, both PML-RAR (S) and (L)
stimulated ATRA-mediated transactivation more strongly than
RAR,13,15 whereas others found that PML-RAR activated
weakly or not at all.170 Some of these differences may have
been due to the use of different cell types or reporter genes. In
general, when coexpressed with RAR, the fusion proteins behaved in a
dominant negative fashion, reducing activation to the level of
PML-RAR alone.157,292 RAR and PML-RAR, although
activating transcription to different extents, had a similar
ED50 for ATRA (~5 × 109 mol/L) and had similar dissociation
constants for ATRA (~1010
mol/L).170,290 Hence, the altered transcriptional activity
of the PML-RAR fusion protein is not related to impaired ability to
bind ATRA. However, there are some subtle differences among the
PML-RAR isoforms in terms of ATRA binding. The PML(S)-RAR isoform
had a higher affinity for 9-cis retinoic acid than
PML(L)-RAR.291 A functional consequence of this effect
was that PML(S)-RAR activated a reporter gene at lower
concentrations of 9-cis retinoic acid than the long isoform.
Furthermore, 9-cis retinoic acid was found to be more effective than
ATRA in inducing differentiation of APL cells harboring the short
isoform in vitro. These data strongly support the notion that
differences in clinical presentation of APL are a consequence of
PML-RAR variants.
The impaired ability of the PML-RAR protein to activate certain
promoters is not due to deletion of the A (activation) domain of
RAR. In parallel transfection experiments, a  A-RAR mutant activated transcription to a similar extent as wild-type
RAR.170,292 However, nuclear receptor A/B domains are
known to act in a cell-type specific manner, and deletion of this
moiety could still explain some aspects of PML-RAR
function.42 Impaired activation is probably due to the
ability of PML-RAR to bind the corepressors SMRT and N-CoR more
tightly than wild-type RAR, requiring pharmacologic doses of ATRA
(106 mol/L) for disassociation. This is in marked
contrast to the physiologic heterodimer of RAR-RXR, which releases
corepressors at 109 mol/L ATRA, potentially
explaining the need for high doses of ATRA to stimulate differentiation
in APL.293-296 The chimeric protein had increased affinity
for SMRT and N-CoR, despite the fact that neither corepressor bound to
PML in vitro or colocalized in vivo, suggesting that fusion of PML to
RAR might modify the E (ligand and corepressor binding) domain of
RAR in an allosteric manner. Underlining the central role of the E
domain of RAR, PML-RAR mutated in the ligand binding site was
unable to release SMRT, even in the presence of large amounts of
ATRA.295
The corepressors N-CoR and SMRT are part of a multiprotein complex that
includes histone deacetylases (HDACs). Deacetylation of histones alters
the conformation of chromatin and its accessibility to the
transcriptional machinery, resulting in transcriptional silencing.46 Binding of ligand to RAR leads to the
release of corepressors and binding of coactivators, many of which may acetylate histones. However, PML-RAR may retain the deacetelyase complex, moderating the ability of the protein to activate
transcription. In fact, PML-RAR associates with HDAC1 (histone
deacetylase 1),295,296 and this may be the basis of the
block of critical myeloid gene expression at physiological doses of
ATRA. The association of PML-RAR with N-CoR, SMRT, and HDAC1 was
further characterized by demonstrating that a fusion protein mutated in
the CoR-box (corepressor binding motif) was both unable to associate
with HDAC1 and block differentiation of transfected
cells.296 Sodium butyrate297 or trichostatin A
(TSA),298 inhibitors of histone deacetelyation, stimulated
ATRA-mediated transcription by PML-RAR,294-296,299 possibly by blocking the activity of residual bound deacetylase complexes. This could explain the synergism of sodium butyrate and ATRA
to accelerate differentiation of APL.294,299,300 In fact,
when exposed to TSA, a resistant NB4 cell line containing PML-RAR
with the E domain mutation mentioned above was able to partially
differentiate in response to ATRA,295 presumably by augmenting the function of endogenous wild-type RAR. TSA was able to
restore ATRA-induced transactivation of an RARE-containing reporter in
these same resistant cells and could potentiate differentiation and
expression of RAR target genes in transfected U937
cells.295,296
PML-RAR also affects other transcriptional pathways important for
myeloid differentiation. ATRA and RAR can inhibit transcriptional activation by the AP1 protein, possibly by a competitive effect between
the ligand-engaged receptor and either fos or jun for a limiting amount
of a common coactivator protein p300 and/or CBP.59,73
Somewhat paradoxically, PML-RAR, when coexpressed with fos and jun,
stimulates transcription from an AP1 binding site containing reporter
gene in the presence of ATRA.301 Whereas PML could not be
shown to directly interact with fos or jun, PML could be detected in an
AP1 DNA-protein complex.203
PML-RAR inhibits transcription by other nuclear receptors. In EMSA
assays, PML-RAR competes with VDR for RXR and prevents formation of
the VDR/RXR DNA-protein complex. In cotransfections assays, PML-RAR
blocked vitamin D3-mediated transcriptional activation, an effect
reversed by the overexpression of RXR.171 PML-RAR also
blocked ligand-mediated activation by the peroxisome
proliferator,287 likely by a similar mechanism of
sequestration of coactivators. In contrast, PML-RAR, like PML,
stimulated transcription mediated by the PR.212 The
mechanism of this activity is obscure, but this finding underscores the
pleiotropic effects of PML-RAR. Lastly, whereas RAR and STAT1
synergistically stimulated transcription from an IFN response
element-containing reporter, PML-RAR did not, suggesting that the
cross-talk between IFN and retinoid signaling may be defective in
APL.120
Distillation of these studies shows several points. (1) The
transcriptional activity of PML-RAR varies depending on cell type
and target promoter. (2) PML-RAR tends to suppress transcription of
RAR target genes at physiological concentrations of ATRA to a
greater extent than wild-type RAR. This effect, due to aberrrant interactions with corepressors, may be critical for differentiation block in APL. (3) On some promoters, PML-RAR can activate
transcription to a similar or greater extent than wild-type RAR at
106 mol/L ATRA. (4) PML-RAR can suppress
ATRA-mediated transcription by endogenous RARs as well as transfected
wild-type receptors. This may be due to competitive binding by
PML-RAR, which on some promoters displays intrisically lower
trans-activation potential, as well as competition with RAR
for coactivators. (5) The synergistic effects of butyrate or TSA and
ATRA on transcription by PML-RAR and in differentiation are likely
due to inhibition of histone deaceteylation and alterations of
chromatin stimualting activation of RAR targets. (6) PML-RAR also
effects the transcriptional function of other nuclear receptors as well
as other transcription factors such as AP1 and STATs.
| |
PML-RAR AND RETINOID RESISTANCE |
ATRA-resistant APL cell lines derived by x-ray
mutagenesis302 were found to lose expression of the
PML-RAR protein, although the fusion gene and mRNA
remained.302 This was due to a protease activity that
degrades exogenous PML-RAR and is blocked by chemical inhibitors of
the proteosome.303 This highlights the importance of the
PML-RAR protein both in generating the APL phenotype and in
mediating the unique sensitivity of the disease to ATRA. These data
also imply that there are secondary changes in APL cells that maintain
the transformed phenotype even in the absence of stable expression of
the protein.
Resistant APL cells were also generated by prolonged culture of NB4
cells121 in the presence of ATRA.304,305 One
such resistant cell line expressed the PML-RAR protein but had an
abnormal retinoic acid binding profile305 and failed to
upregulate tissue glutaminase II expression in response to ATRA. This
was due to a missense mutation in the E domain of
PML-RAR,306 which abolished its ability to bind ligand
and mediate trans-activation by ATRA. These data support the
notion that PML-RAR has a critical effect in blocking gene
expression at low doses of ATRA and further underscores the fact that
therapeutic response to ATRA in APL is dependent on the ability of the
chimeric protein to activate transcription in the presence of ligand,
reversing the blockade of target genes. In this and other resistant
cell lines, at least one gene, CD18, continued to be induced by ATRA.
This indicates that the remaining endogenous RARs within the cell can
activate a subset of target genes, but the genes most critical for cell
differentiation continue to be inhibited by PML-RAR.
These types of mutations are clinically relevant, because recent
studies showed mutations in the ligand binding domain or adjacent AF-2
region in nearly 15% of patients both de novo but particularly after
prolonged ATRA treatment,307,308 indicating that ATRA
treatment puts a strong selective pressure for clones with defects in
PML-RAR.308 However, the sequence of PML-RAR in cell
lines derived from resistant patients and in primary patient specimens
is most frequently normal, indicating that another mechanism must play
a major role in ATRA resistance, such as loss of the PML-RAR fusion
protein due to accelerated degradation or activation of novel
oncogenes.303,308,309
| |
PML-RAR AND THE NUCLEAR BODY |
Whereas PML is localized in 6 to 30 large nuclear bodies/cell measuring
between 0.2 and 0.3 µm191 in APL, it is delocalized to
greater than 100 small (0.1 µm) microspeckles197 due to
the ability of PML to heterodimerize with PML-RAR though the
coiled-coil motif.170,191 PML-RAR draws other nuclear
proteins, including SP100,198 PLZF,216
RXR,197 and Rb,229 into the microspeckled structure as well. These microspeckles have no evident
structure198 and colocalize with nascent RNA, signifying
the transcriptional function of PML-RAR.191,201 In at
least one set of studies, a large proportion of PML-RAR fusion was
localized in the cytoplasm rather than the nucleus.173 This
is consistent with the notion that PML-RAR, under low physiological
concentrations of retinoids, acts as a dominant negative receptor
drawing critical factors away from loci controlled by RAR to a new
set of loci or to a transcriptionally inactive compartment.
ATRA treatment of APL cells relocalizes the PML protein into the
wild-type nuclear body configuration.173,191,197,198,310 This is largely due to degradation of
PML-RAR95,96,191,311,312 through the action of the
proteosome,303 likely by the induction of a caspase 3-like
activity after ATRA treatment.313 The specific cleavage of
PML-RAR occurs C-terminal to the the RING, B-boxes, and coiled-coil
motifs of PML, yielding a product recognized by RAR antibodies that
contains residual PML sequences.95,96 The resulting protein
could be predicted to be unable to bind wild-type PML, which would then
be released and free to form its usual macromolecular complex in the
NB. The remaining truncated PML-RAR protein might function in a
similar fashion as wild-type RAR, activating its target genes and no
longer sequestering other proteins critical for cell differentiation
through the N-terminal PML moiety. However, early after ATRA treatment,
reactivity to RAR antibodies is detected in the large nuclear
bodies, suggesting that the PML-RAR protein itself undergoes some
conformational shift or novel protein association after ATRA treatment,
which then allows it to colocalize with wild-type PML in the
NBs.173,197,198 It should also be noted that the
PML(S)-RAR isoform does not contain the sequences required for
caspase cleavage and does not undergo degradation after ATRA, yet these
patients respond to ATRA therapy.313,314 In addition, in a
model system, ATRA could induce differentiation even in the presence of
caspase inhibitors, suggesting that the degradation of PML-RAR may
not be essential for therapeutic response.313 The
relatively slow reorganization of the NB is accelerated by other
agents, such as cyclic AMP,310 which also increase the rate
of differentiation, suggesting that altered phosphorylation of the
PML-RAR fusion changes its rate of degradation and/or
relocalization. Thus, PML-RAR relocalization is highly correlated
with induction of differentiation in APL cells, suggesting that
disruption of some component of the nuclear body other than PML plays a
key role in this process.
Other agents promote normalization of the nuclear body structure
through different mechanisms. Recent studies indicate that arsenic
trioxide (As2O3), a component of traditional
Chinese medicine, induces complete clinical remission of APL in ATRA
resistant patients.315 Whereas reaggregation of the nuclear
body takes 1 to 2 days of ATRA exposure, treatment with
As2O3 leads to rapid formation of wild-type
pattern nuclear bodies within 6 hours, followed by loss of PML staining
after 24 hours.311,316,317 This effect was observed both in
NB4 and other cell lines and was enhanced by IFN.179,311 During this process, both PML and PML-RAR are targeted to the nuclear body and then rapidly degraded, although there is no effect on
other NB proteins such as SP100 and relatively little degradation of
endogenous RAR.318,319 Furthermore,
As2O3 increased the transfer rate of PML from
the nucleoplasm to the nuclear matrix179,311 and increased
PML levels within the NB, accelerating apoptosis.252 It was
recently shown that As2O3 induces the
phosphorylation-dependent covalent linkage of PML to the ubiquitin-like
molecule sentrin, perhaps targeting the protein for degradation. The
C-terminal portion of PML was required for its targeted degradation.
Whether sentrin binds PML-RAR is controversial and whether
sentrinization or ubiquination of PML-RARa is required for its
degradation is not yet certain.180,227
As2O3 induces degradation of PML-RAR even in
APL cell lines resistant to ATRA.318-321 Both in vitro and in cells derived from patients undergoing As2O3
treatment, this correlates with only partial differentiation of the
malignant promyelocytes and predominantly the induction of
apoptosis.311,318-320 Simultaneous treatment with ATRA and
As2O3 enhanced differentiation and apoptosis of
NB4 cells319 and enhanced survival of animals harboring APL
(H. de Thé, personal communication, December 1998). However, this
was not the case in fresh human APL cells318 treated in
vitro, making it not yet certain whether ATRA and arsenic might best be
used concommitantly or as sequential agents in the treatment of APL.
The organic arsenical melarsoprol may also provide effective treatment
of APL321 and other hematological
malignancies.322 As2O3 and
melarsoprol induce apoptosis of an APL cell line without detectable
PML-RAR as well as PML/ murine
fiboblasts.321 However, others have found a
dependence of PML expression for arsenic induced apoptosis (H. de
Thé, personal communication, December 1998), and one group found
that a cell line harboring PLZF-RAR could not be induced to undergo
apoptosis with arsenic,227 suggesting that the extreme
sensitivity of APL cell lines to arsenic may be due to their dependence
on PML-RAR for continued growth and the prevention of apoptosis.
Intriguingly, antimony, a metal in the same column of the periodic
table as arsenic, can also induce the degradation of PML
and induction of apoptosis of APL cells,323 suggesting a
common mechanism of covalent modification of critical cellular proteins by these heavy metals.
These results lead to a model in which treatment of APL can occur by
two different mechanisms, each rescuing the disrupted nuclear body and
perhaps restoring a growth control mechanism to the cell. In both
cases, degradation of PML-RAR releases the complete block on cell
differentiation. After both ATRA88 and As2O3315 treatment, an initial
hyperleukocytosis is noted. However, the complete lack of PML-RAR
after As2O3 treatment leads to apoptosis, whereas the residual PML-RAR fragment present after ATRA treatment, in combination with residual RAR, induces the genes critical for
cell differentiation. How these agents cause the retargeting of
PML-RAR to the nuclear body is unknown.
As2O3 can react with sulfhydryl groups and
alters phosphorylation pathways, a fact supported by the finding that
As2O3 treatment is associated with hyperphosphorylation of the RAR itself.311 ATRA may
cause a conformational shift in PML-RAR, allowing new sets of
proteins to interact with the ligand binding domain. RXR may play a
role in this conformation shift as well. When treated with ATRA plus an
RAR antagonist, APL cells did not differentiate or reorganize their
NBs. However, when treated with the same RAR antagonist plus a RXR
agonist, both NB reorganization and differentiation ocurred.68 These experiments underline the complexity of
retinoid signaling and highlight the importance of both components of
the RAR/RXR heterodimer in myeloid differentiation and APL pathogenesis.
Although the disruption of the nuclear body in t(15;17) APL is one of
the most dramatic features of this disease, it may not be absolutely
required for the pathogenesis of APL, because PML is in the wild-type
configuration in variant forms of APL. Hence, it may be the degradation
of PML-RAR rather than changes in PML/NB function that may be most
critical for the induction of differentiation in APL. Alternatively, a
component of the NB other than PML may be sequestered by all of the
RAR chimeras.
| |
CELLULAR MODELS OF PML-RAR FUNCTION |
Cellular models of PML-RAR function have been hampered by the
toxicity of the fusion protein, as underscored by studies in which the
PML-RAR fusion protein could not be expressed after retroviral
infection in nonhematopoietic cell lines and was expressed in only a
few hematopoietic cell lines. PML-RAR retroviruses were difficult to
generate due to the growth-suppressive effects of the protein on
fibroblasts, including retroviral packaging cell
lines.324-326
The most successful model of PML-RAR function in APL was constructed
in the monocytoid U937 cell line.324 Cells stably or inducibly expressing PML-RAR failed to differentiate in response to
ATRA or a combination of vitamin D3 plus transforming growth factor (TGF).324,327 Under physiological
concentrations of ATRA (109 mol/L), PML-RAR
expression was associated with an increase in cell growth rate.
However, when treated with 106 mol/L ATRA,
PML-RAR expression was associated with markedly decreased cell
proliferation and increased differentiation. The change in growth rate
was due to an increase in apoptosis and not to alterations in the cell
cycle. In addition, when grown under conditions of reduced serum,
PML-RAR-expressing U937 cells proliferated, whereas control cells
underwent apoptosis. PML-RAR also blocked apoptosis in response to
TNF.328 TNF resistance was due to a posttranscriptional
downregulation of the TNF receptor, allowing the APL cell to escape an
autologous growth inhibitory mechanism, because APL cells secrete high
levels of TNF.329 From these data it was proposed that
PML-RAR might function to promote cell survival. This idea was
supported by the fact that myeloid, G-CSF-dependent TF-1 cells
expressing PML-RAR were protected from apoptosis induced by G-CSF
withdrawal.314,330 In addition, recent data indicate that
ablation of PML-RAR expression in NB4 APL cells either by homologus
recombination or expression of a ribozyme induces
apoptosis,331,332 even in ATRA-resistant cells. The
fraction of cycling cells in APL is relatively low; thus, the
persistence of cells due to the anti-apoptotic effects of PML-RAR
could be critical. If PML-RAR prevents apoptosis when cells are
grown under physiological conditions of ATRA, how does the protein
encourage apoptosis when such cells are treated with phamacological
ATRA or As2O3?311,315,318,319,333
One possibility is that PML-RAR is degraded under these
conditions,96,311 removing the protective agent.
Alternatively, these agents, through the action of PML-RAR, may
activate novel target genes that play a role in cell cycle arrest and
apoptosis. The p21 gene is such a candidate, being activated by both
ATRA130 and arsenic.319 As2O3-mediated apoptosis of APL cells is
accelerated by agents that deplete cellular glutathione, a scavanger of
free radicals.334 Given recent data that p53 induces
apoptosis through generation of reactive oxygen species,335
there may be overlap between the p53 and As2O3
apoptotic pathways.
One of the drawbacks of the U937 model is that these cells undergo
monocytic rather than granulocytic differentiation. One model, possibly
more reflective of the pathophysiological role of PML-RAR, was
created by transient expression of PML-RAR in HL60 cells. This
inhibited granulocytic differentiation induced by ATRA and vitamin D3
but not granulocytic differentiation induced by dimethyl sulfoxide
(DMSO) or monocytic differentiation induced by phorbol
ester. These results support a relatively specific mechanism of action
for PML-RAR upon nuclear receptor pathways. However, NB4 cells
cannot differentiate in response to vitamin D3,300,327 but
when ATRA and D3 are added in concert or sequentially (ATRA first),
marked differentiation and inhibition of proliferation occurs. In
addition, NB4 cells are resistant to polar compounds such as sodium
butyrate or HMBA, unless pretreated with ATRA for a period as brief as
30 minutes.300 Thus, PML-RAR may affect non-nuclear
receptor differentiation pathways as well (see below). Recently,
PML-RAR was shown to enhance the proliferation of murine bone marrow
progenitor cells after retroviral transfer, allowing these cells to be
serially replated ex vivo. However, the cells remained growth factor
dependent, suggesting that PML-RAR on its own cannot completely
transform the cell. The cell lines resulting from PML-RAR expression
were undifferentiated and could be induced to stop proliferation and
undergo differentiation after ATRA treatment.336
The U937 model was used to determine which structural features of the
PML-RAR fusion protein were critical for effects on cell growth and
differentiation.163 PML expression did not block differentiation induced by vitamin D3 and TGF, whereas RAR did slightly, perhaps due to sequestration of RXR. PML-RAR was a more
potent inhibitor of differentiation. Deletion analysis showed that the
first coiled-coil motif of PML-RAR was required for its ability to
block differentiation, but deletion of this segment did not affect
PML/PML-RAR interaction, NB disruption, or RARE-dependent trans-activation. This suggests that disruption of PML within the
nuclear body is not critical for the action of PML-RAR and that the
ability of PML-RAR to interact with an unidentified factor, through
the coiled-coil motif, may be critical for its function. In support of
this hypothesis, deletion of coil 2 prevents PML-RAR from
delocalizing PML from the NBs, but still allows disruption of the
nuclear body pattern of Sp100. This protein or another of the multiple
proteins within the nuclear body such as Rb229 could
represent the target protein of PML-RAR. This is not likely to be
RXR, because coexpression of RXR prevented differentiation block
induced by wild-type RAR but not the block mediated by PML-RAR.
The RING finger and B box motifs are also always found in the
PML-RAR fusion protein of APL patients, suggesting that the
integrity of the RBCC unit is required for leukemogenesis. Furthermore,
the ability of PML-RAR to induce apoptosis in nonhematopoietic cells
depends on the integrity of the RING finger/B-box motifs and the
microspeckled localization of PML-RAR. How this relates to APL
pathogenesis by PML-RAR is unclear.326 Lastly, as noted, there are subtle differences in the PML(L)-RAR and PML(S)-RAR isoforms. In TF-1 cells, only the long PML isoform inhibited cell growth, whereas only the short isoform protected the cells from growth
factor withdrawal. When cells were treated with ATRA, they underwent
apoptosis, with the long isoform showing a more prominent effect.314
Several major conclusions can be reached from these cellular models.
(1) PML-RAR does not activate cell growth or confer factor-independent growth to cells and in this regard is not a conventional oncogene. (2) PML-RAR alters the cell setpoint for apoptosis. It inhibits apoptosis due to growth factor withdrawal in
hematopoietic cells, but encourages it in the presence of ATRA or
arsenic. In many nonhematopoietic cells, PML-RAR is toxic and
induces apoptosis. (3) PML-RAR can inhibit differentiation mediated
by nuclear receptor pathways and some non-nuclear receptor pathways.
The mechanism of cross-talk with nonreceptor pathways is unknown. (4)
PML-RAR requires the first coiled domain of PML and the AF2 domain,
which binds HDAC complexes, of RAR to block differentiation. The
second coiled domain, which delocalizes PML, is dispensible for
differentiation block. The first coiled-coil motif of PML may contact a
critical cofactor. (5) The PML(S)-RAR isoform, which lacks
phosphorylation sites, and the nuclear localization sequence of PML may
have somewhat different biological properties from the PML(L)-RAR isoform.
| |
ANIMAL MODELS OF PML-RAR FUNCTION |
Several laboratories have tried to model APL in animals. Infection of
avian bone marrow with a PML-RAR-containing retrovirus resulted
in an undifferentiated form of leukemia that did not respond to ATRA.337 Curiously, the PML-RAR gene harbored
in these leukemic clones bore two point mutations. The first, located in the RING finger, caused the fusion protein to lose the typical microspeckled pattern and have altered transcriptional properties. The
second replaced a serine with an alanine residue in the coiled-coil domain, causing the loss of a potential phosphorylation site. These
studies, although successful in demonstrating the oncogenic potential
of PML-RAR, highlight the difficulties of working with this toxic
protein.326 Interestingly, infection of mouse bone marrow
with a C-terminal truncated form of RAR also yields the outgrowth of
a lymphohematopoietic precursor cell, indicating that there may be two
points of control by ATRA in hematopoiesis, one at the pluripotent
progenitor stage and a second at the promyelocyte stage.338
Why RAR disruption clinically yields only the promyelocyte phenotype
is uncertain.
The first transgenic mouse created that expressed the PML-RAR fusion
used the CD11b promoter, which was expressed relatively late in myeloid
maturation. These mice did not develop APL or a preleukemic syndrome,
but did show a defect in myeloid precursor response to cytokines and
profound neutropenia after sublethal irradiation, implying that the
PML-RAR protein impaired myeloid development.339 Another
transgenic murine model expressed the PML-RAR transgene from the
metallothionine promoter.340 The investigators had
difficulty obtaining transgenic founders, likely reflecting the poor
tolerance of PML-RAR by nonhematopoietic cells. One animal line
expressed PML-RAR only in the liver and brain, which was induced by
the addition of Zn2+ to the animals' drinking water. These
animals developed liver pathology, including hepatocellular carcinoma
after only 5 days of induction with zinc. ATRA treatment did not
prevent the emergence of liver disease, which was associated with an
increased proliferation rate and no change in spontaneous apoptosis.
These experiments confirmed the oncogenic nature of PML-RAR and
suggested that, under certain circumstances, the protein could
accelerate cell proliferation. Two groups performed transgenic
experiments with PML-RAR using the cathepsin G
promoter.341,342 These mice developed a preleukemic
syndrome characterized by an increase in immature myeloid forms in the
bone marrow and splenomegaly due to extramedullary hematopoiesis.341 About 10% to 30% of the animals
developed leukemia, with a median latency of 300 days; it was also
associated with anemia, thrombocytopenia, and massive splenomegaly. A
modest peripheral promyelocyte count of only 7% was
noted,342 with some cells harboring Auer rods. No bleeding
diathesis was noted. Unlike human APL, many fully differentiated
granulocytes were present in the peripheral blood before treatment with
ATRA. ATRA treatment led to an initial increase in peripheral white
blood cell count, reminiscent of the retinoic-acid
syndrome88 and consistent with the mobilization of cells
from the marrow. This was followed by a decrease in the leukocyte count
and the appearance of differentiated neutrophils. However, after ATRA
treatment, Grisolano et al341 found that promyelocyte
counts decreased and the cells appeared to undergo apoptosis rather
than differentiation. Therefore, whether ATRA induced differentiation
of the promyelocytes or selectively killed the immature cells is not
certain, because differentiated cells were present both before and
after ATRA treatment. These animals therefore offer a somewhat
imperfect model for differentiation therapy. Intriguingly, as in
humans,7,8 all mice relapsed even with the continuation of
ATRA, suggesting that other oncogenic lesions must be involved in APL
development.299
The mouse model of APL most similar to human disease was generated by
use of the MRP8 promoter, which is expressed at the promyelocyte to
metamyelocyte stage and continues to be active in mature
neutrophils,343 as opposed to the cathepsin G gene, which
is expressed during a more narrow window of promyelocyte differentiation.344 These mice also developed a preleukemic
phase, and about one third developed promyelocytic leukemia with a
median latency of 6 months345 (S. Kogan, personal
communication, July 1998), accompanied by bleeding, anemia,
thrombocytopenia, and a low leukocyte count, all characteristic of
human APL. When these cells were placed into culture and treated with
ATRA, differentiated neutrophils were observed. The transgenic mice
also developed epidermal papillomas, which further demonstrated the
neoplastic activity of the PML-RAR fusion. When the animals were
treated with ATRA, mature neutrophils appeared in the peripheral blood and marrow and splenomegaly was reduced, consistent with clinical differentiation. Highly purified, residual nonleukemic progenitor cells from APL patients are PML-RAR negative,346
suggesting that the PML promoter driving PML-RAR expression must
function at a specific stage of myeloid differentiation. Therefore, a
knock-in347 strategy for the creation of transgenic mice
might yield the most physiologic model of t(15;17) translocation APL.
In all of these models, the delay in onset of leukemia suggests that a
second, as yet uncharacterized, genetic hit is required for neoplastic
transformation. Whether the leukemias that do develop in the mice are
monoclonal or polyclonal has not yet been determined. A monoclonal
origin would be consistent with a model of tumor promotion induced by
PML-RAR, potentially by preventing apoptosis and blocking
differentiation, followed by a second genetic lesion. Such a multistep
pathway was modeled by the introduction of N-ras into the hematopoietic
cells derived from mice harboring the PML-RAR fusion expressed from
the CD11b promoter.348 As a result, there was a 10- to
100-fold synergistic increase in myeloid colonies. With this proof of
principle, we can anticipate experiments in which PML-RAR mice could
be crossed with mice expressing activated oncogenes or lacking tumor
suppressors. The APL that develops spontaneously in PML-RAR mice
could also be screened for secondary mutations required for pathogenesis.
| |
SUGGESTED MODEL OF PML-RAR ACTION IN APL |
Myeloid differentiation usually occurs at physiological levels of ATRA
(108 mol/L), which activates key RAR target
genes. These target genes may contain high-affinity RAREs or bind
factors that cooperate with the RAR to load the basal
transcriptional machinery onto the promoter, even when only a fraction
of cellular RAR is engaged by ligand. The highly expressed
PML-RAR protein may functionally sequester RAR cofactors from the
wild-type receptor or bind to critical genes in place of RAR. Even
if a PML-RAR/RXR heterodimer bound to the critical
genes, the increased affinity of PML-RAR for
corepressors294,299 would make it a poor activator of
RAR target genes. This would overwhelm the usual tonic effect of
RAR to induce myeloid differentiation at low ATRA concentrations
(Fig 5).
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| Fig 5.
Suggested model of PML-RAR action in APL. (A) At
109 to 108 mol/L ATRA, PML-RAR
prevents activation of key target genes required for myeloid
differentiation by sequestration of RXR and other RAR cofactors,
inhibiting normal RAR function. In addition, PML-RAR may bind to
RAR targets as a homodimer or as a heterodimer with RXR and inhibit
transcription of these genes by recruitment of corepressor/histone
deacetlyase complexes. PML-RAR also may affect
transcription mediated by AP1 and IFN-responsive factors and can
sequester PLZF and potentially affect its function. PML-RAR prevents
apoptosis through unknown mechanisms and delocalizes PML and other
proteins from the nuclear body, although the importance of this is
uncertain, because the NB is normal in the other forms of APL. (B) In
the presence of pharmacological doses of ATRA, the PML-RAR fusion is
degraded and releases PML and other cofactors. The NB structure is
restored. A residual fragment of the PML-RAR fusion and/or the
wild-type RAR, which is upregulated in response to ATRA, can then
stimulate transcription of myeloid target genes. The blockade of other
signaling pathways is released and the anti-apoptotic effect of
PML-RAR is lost. As a result, terminal cell differentiation can
proceed.
|
|
In the presence of pharmacological doses of ATRA, the PML-RAR fusion
releases the corepressors and stimulates transcription of target genes
that allow myeloid development to proceed. Furthermore, the PML-RAR
protein is degraded95,96 and wild-type RAR is upregulated,94 shifting the balance of power of RARs in the cell from PML-RAR to RAR. However, the fact that PML-RAR can confer ATRA sensitivity to mutant HL60 cell lines without endogenous wild-type RAR suggests that PML-RAR does mediate at least part of
the pro-differentiative349 effect. A PML-RAR/RXR
heterodimer is probably the mediator of differentiation, because this
process is synergistically stimulated by a combination of RAR- and
RXR-specific ligands.68,76 In addition, brief treatment of
NB4 cells or fresh APL cells with ATRA allows potent differentiation of
APL cells to proceed in the presence of other agents such as
hexamethylene bis acetamide (HMBA), cyclic AMP, and vitamin
D3,300,350 suggesting that rapid transcriptional events
before PML-RAR degradation mediate differentiation. These events
might also include activation of other nuclear receptors, STATs and
AP1. In contrast, arsenic degrades PML-RAR fusion without
RAR-mediated signaling. Modest differentiation might occur in this
case by low level signaling through the endogenous RAR. A component
in the pathogenesis of APL may include the delocalization of one or
more key proteins from the NB. Current evidence points away
from this being PML itself.
| |
RAR-PML |
The reciprocal RAR-PML fusion generated in
t(15;17)11,351 is present in 70% to 80% of APL
cases351,352 (Fig 3). Two transcripts can be generated from
the alternative RAR promoters, of which RAR1-PML was the most
common.353 As in the case of PML-RAR, several different
forms of RAR-PML were also found due to alternative breakpoints
within the PML gene. RAR1-PML and RAR2-PML transcripts from
patients with breakpoints 5' in the PML gene encode RAR-PML proteins, whereas fusion transcripts derived from a more 3'
breakpoint in the PML gene only encode a truncated peptide containing a
portion of the RAR A domain. RAR-PML contains the A1 or A2 domain of the RAR protein fused to a variable portion of the PML protein, due
to alternative splicing, including the serine-rich C-terminal domain.
It is hard to predict the effects of RAR-PML, because the role of
the C-terminus of PML is unknown. There were cases of APL associated
with interstitial, nonreciprocal fusions of the PML and RAR genes
that did not generate an RAR-PML fusion gene.262-264
Furthermore, there is no difference in ATRA sensitivity or clinical
outcomes of patients who do or do not harbor the RAR-PML transcript.352,353 Patients with prolonged remissions of
APL may express the RAR-PML transcript and not the PML-RAR
transcript, suggesting that the small number of cells that harbor these
genes may not have leukemogenic potential,354 although a
sole APL patient was reported to posses the RAR-PML and not the
PML-RAR transcript.355 In general, RAR-PML does not
appear to be required for the pathogenesis of APL. Although transgenic
mice harboring the RAR-PML fusion did not develop leukemia, when
crossed with PML-RAR mice, leukemia developed with greater
frequency.356 Hence, the RAR-PML may contribute to the
disease process.
| |
PLZF |
The PLZF gene.
The PLZF gene was initially identified by its rearrangement in an APL
patient from Shanghai with translocation
(11;17)(q23;q21)357-359 (Fig 6). Eight confirmed
cases of t(11;17)(q23;q21) APL fusing the PLZF and RAR genes were
described,360-363 and a recent workshop identified a total
of 8 cases around the world.364 Morphologic review of the
original 6 patients and the 8 cases of the workshop showed features
intermediate between M2 and M3 leukemia, with sparser granules, a lack
of Faggot cells, and the absence of a bilobed nucleus.361
Strikingly, these 6 patients were resistant to differentiation therapy
with ATRA as well as chemotherapy. Leukemic cells from these patients
could not be induced to differentiate with ATRA in
vitro.360,361 Translocation (11;17)(q23;q21)-APL is unique
in its resistance to ATRA, suggesting a critical difference between
PLZF and the other four fusion partners of RAR in APL (Table 4). However, in
opposition to this view, cells from 1 patient with t(11;17)(q23;q21)
APL could be induced to differentiate when treated with a combination
of ATRA and G-CSF but not ATRA alone,365 and this patient
was successfully treated with a combination of ATRA and
G-CSF.366 One other patient achieved a remission with
concurrent ATRA and chemotherapy,363 suggesting that the resistance of this form of APL may not be absolute.
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| Fig 6.
Functional domains of the PLZF protein and structure of
the PLZF-RAR and reciprocal RAR-PLZF proteins generated in
t(11;17)(q23;q21) APL. PLZF-RAR always contains the N-terminal
POZ/BTB-self-association domain. Heterogeneity in the PLZF gene
breakpoint can yield PLZF-RAR fusion proteins, including either the
first two or first three zinc fingers of PLZF. The reciprocal
RAR-PLZF protein can bind to PLZF binding sites and contains the
last six or seven of the nine PLZF zinc fingers linked to the AF1
activation domain of RAR rather than the PLZF repression domains.
|
|
The PLZF gene, localized on chromosome 11q23, yields a 7-kb mRNA that
codes for a zinc finger transcription
factor.289,359,367,368 The PLZF gene is 1 Mb telomeric to
the MLL/HRX gene, which is frequently rearranged in
leukemia.369-371 The genomic structure of PLZF is
incompletely known, but it is clear that the N-terminal portion is
encoded on a single exon and that the C-terminal zinc finger motifs are
encoded by a number of small exons (Baysal et al372 and Z. Chen, personal communication, June 1998). PLZF codes for a
DNA-binding protein of 673 amino acids with nine Krüppel-like C2H2 zinc finger domains with a predicted
molecular weight of 74 kD (Fig 6) that migrates through polyacrylamide
gels with an apparent MW of 80 to 90 kD.
The N-terminal 118 amino acids encode a POZ (Pox virus and Zinc finger)
or BTB (Broad Complex, tramtrack, Bric a Brac) domain. The
POZ/BTB domain mediates protein self-association and heterotypic associations373 and acts as a transcriptional repression
domain within zinc finger374-377 and
basic-zipper378 transcription factors. The POZ/BTB domain
can be involved in chromatin remodeling and histone
mobilization379,380 and transcriptional repression through interaction with histone deacteylase (see below). Physical
characterization of the PLZF POZ/BTB domain found it to be a tight
dimer in solution (Kd = 2 × 107), with a high
amount of helical structure.381 Characterization of a 1.9Å
crystal structure of the BTB/POZ domain of PLZF382 confirmed these findings, showing a highly intertwined dimer with a
large hydrophobic interface. The top portion of the dimer structure forms a groove exposed to solvent lined with conserved charged amino
acids potentially representing a peptide binding site (Fig 7). Several
missense mutations of conserved residues interfere with dimer contacts
and disrupt the ability of the POZ domain to repress transcription,
suggesting that dimerization and repression may be closely
linked.605
| |
PLZF NUCLEAR LOCALIZATION |
In transfected cells, PLZF is localized to the nucleus and
is289,368 phosphorylated on serine and threonine residues
(Shaknovich and Licht, unpublished data). MDS, a primitive leukemic
cell line, expresses high levels of PLZF mRNA and PLZF protein
when treated with the calcium ionophore A23187.289
Confocal microscopy of MDS cells showed that PLZF localized to
approximately 50 small nuclear subdomains, with a lower level of
diffuse nuclear staining also noted, whereas only 10 PML-containing NBs
were noted per cell. The speckled pattern of subnuclear expression of
PLZF depends on the presence of the POZ/BTB domain as removal of this
domain leads to expression in a diffuse nuclear pattern.383
Given the potential importance of the expression of the PML protein in
the NB, it became important to determine whether PLZF was expressed in
the same or similar domains. In hematopoietic cells that naturally express both PML and PLZF as well as in transiently transfected nonhematopoietic cells, wild-type PML and PLZF could colocalize in
nuclear body structures. However, this colocalization was not complete,
indicating that PLZF may act in both the nuclear body and other
subnuclear compartments.216 The PLZF-RAR fusion protein, either transfected into cells or naturally expressed in blasts from a
patient with t(11;17)(q23;q21) APL, did not colocalize with PML or
delocalize PML from nuclear body structures.216 This critical fact indicates that delocalization of the PML component of the
nuclear body is not required for the pathogenesis of APL. In contrast,
in t(15;17) APL cells, PLZF is delocalized into a microspeckled pattern
identical to PML-RAR.216,299 The coiled-coil region of
PML, which is also responsible for PML self-association, was required
for the PML-PLZF interaction. The first two zinc fingers of PLZF and
not the POZ/BTB domain mediates the interaction of PLZF with PML (A. Zelent, personal communication, December 1998). Therefore, PML and
other related RING finger proteins might be cofactors for PLZF
function. Delocalization of PLZF might be a common and central theme in
the pathogenesis of APL, because PLZF is also found in an abnormal
microspeckled pattern and in t(5;17)-APL associated with the NPM-RAR
protein.384
| |
PLZF EXPRESSION |
PLZF mRNA is expressed in myeloid cell lines such as KG1, HL60, and NB4
as well in as the multipotent cell line FDCPMixA4. It is expressed at
lower levels in more differentiated erythroleukemia, promyelocytic, and
monocytic cell lines359,368 as well as in peripheral blood
mononuclear cells,359 tissue macrophages (Fallon and Licht,
unpublished data), and pro-B-cell lines.368
PLZF is also expressed in mature B-cell and chronic lymphocytic
leukemia (CLL) specimens.385 PLZF is
downregulated during ATRA-mediated differentiation of NB4 and HL60
cells (Chen et al359 and Chen et al, unpublished
data) and during differentiation of FDCPMixA4. In
contrast, PLZF is upregulated in the MDS cell line after treatment with
calcium ionophore, perhaps recapitulating some aspect of monocyte
development.289 In embryonic stem cells, PLZF levels increase as the cells are allowed to form embryoid bodies that presumably contain hematopoietic elements. Finally, CD34+
human progenitor cells could be immunostained with PLZF antisera in a
distinct nuclear speckled pattern.368 When such cells were placed into culture and allowed to differentiate, PLZF levels transiently increased then declined (C. Labbaye, personal
communication, December 1998). Incubation of human bone marrow with
antisense PLZF oligonucleotides led to a decrease in the number of
burst-forming unit-erythroid (BFU-E) and colony-forming
unit-granulocyte-macrophage (CFU-GM) colonies
(Shaknovich et al, unpublished data). Taken together,
these data indicate that PLZF expression may be important for the
maintenance or survival of hematopoietic stem cells and or early
progenitors. Scheduled, regulated downregulation of PLZF may be
required for normal hematopoietic differentiation and proliferation.
In the murine embryo, PLZF is expressed in the aorta, gonadal,
mesonephros region (AGM), a zone containing hematopoietic precursors. Also, during mouse embryogenesis, PLZF is prominently expressed in the
developing neural tube. Expression throughout the central nervous
system (CNS) is initially uniform at day 8.5 postconception (pc). Subsequently expression is downregulated in
rhombomeric segments 3 and 5 at the same time that genes such as
krox-20 and hoxb2 are upregulated in these
segments.367 A PLZF site was found in the hoxb2
5' flanking region and PLZF could repress the hoxb2 promoter in cotransfection assays,386 suggesting that PLZF
might directly regulate this gene. By 10 days pc, PLZF expression is restricted to the boundaries of the rhombomeres, perhaps acting to
limit the expression of critical pattern formation genes. Early widespread expression of PLZF may initially repress developmental programs within the CNS, and the selective downregulation of PLZF could
lead to segmental identities.387 Similarly, PLZF may
repress the differentiated phenotype of myeloid cells, and its
downregulation may allow differentiation to proceed (see below). It is
reasonable to theorize that altered regulation of homeobox genes may be
central to PLZF action in the development of both the CNS and
hematopoietic system. Other sites of PLZF expression include neural
crest cells, branchial arches, facial processes, and apical epidermal
ridges of the developing mouse and chick limb buds367,387
(C. Tabin, personal communication, July 1998). The latter are sites of
signaling between the epithelium and the underlying mesenchyme and
suggest a role for PLZF in limb patterning. PLZF is also expressed in the mesonephros, a precursor kidney structure, and in the dilated structures found in autosomal dominant polycystic kidney disease (P. Wilson and J. Licht, unpublished data), which are felt to partially recapitulate early renal development. Murine PLZF expression is considerably upregulated in the liver, heart, and kidney in the
perinatal period and shortly after birth. In mutant albino mice that
have a defect in tyrosine metabolism PLZF, along with a number of
liver-specific factors, including HNF-1, HNF-4, and C/EBP, fail to
be induced at birth, grouping these genes in a common regulatory
pathway.367 Lastly, a search of the EST database (http://www.tigr.org) indicates that PLZF is expressed in both skeletal
muscle and adipose tissue. In summary, although isolated by its
involvement in APL, PLZF may play a role in nervous system development
and limb patterning, renal development, hematopoietic development, and
energy metabolism. Preliminary analysis of PLZF knockout
animals388 found that they weighed up to 40% less than heterozygous littermates and have abnormally kinked tails and multiple
skeletal defects, including foreshortening of the limb and fused
digits. This suggests that PLZF, like RAR, might affect the
expression of Hox genes involved in limb and body patterning. Additionally, PLZF may influence genes responsible for apoptosis limb
and digit development. Homozygous PLZF null mice are sterile with
testicular hypotrophy and impaired spermatogenesis. Disruption of other
transcription factors, including CREM, RXR,389 and A-myb,390 has a similar phenotype, suggesting similar or
overlapping functions. Intriguingly, cyclin A, the expression of which
is affected by PLZF (see below), is expressed in a distinct
developmental pattern in the developing testis.391,392 This
suggests that disruption of PLZF expression might affect cell cycle
regulators critical for spermatogenesis. PLZF null mice are viable,
and, to date, these mice have not exhibited an obvious hematopoietic
phenotype and neither have they developed leukemia or other tumors.
This does not rule out a role for PLZF in hematopoiesis and could
indicate the presence of redundant genes to partially compensate for
the lack of PLZF during development.
| |
TRANSCRIPTIONAL FUNCTION OF PLZF |
PLZF is a sequence-specific DNA binding protein that can recognize a
TA-rich sequence derived from a pool of randomized
oligonucleotides.393,394 A binding site for PLZF was
fortuitously discovered in a yeast two hybrid screening experiment.
PLZF fused to an acidic activation domain was isolated by its ability
to activate a bacterial lex operator-containing reporter gene in
yeast.395 The lex operator sequence actually has some
similarity to the artificially selected PLZF site, a PLZF binding site
within the cyclin A promoter, and a human genomic DNA fragment. With
alignment of these sites, a relatively loose consensus sequence of GT
(A/C)(A/C) AGT can be derived. The PLZF binding site derived from site
selection can be recognized by the C-terminal seven zinc fingers
retained in the RAR-PLZF fusion protein. Similarly, the lex operator
site can be bound by proteins containing the last 7 or 5 or 4 zinc fingers of PLZF.386,395 The exact role of the first two
zinc fingers, which are retained in PLZF-RAR in DNA binding, is not yet clear; they may play a role in associations with proteins such as
PML. Given the relatively small length of the PLZF binding site, it is
likely that the last 4 finger motifs may directly bind DNA, whereas
others may either play a supporting role by interacting with the
phosophate backbone in a manner similar to the Gli zinc finger
protein,396 whereas others might participate in protein interactions.
Reporter genes containing either the artificial PLZF binding
site394 or the lex operator294,397 are
repressed by coexpression of the PLZF protein. In contrast, the
RAR-PLZF fusion protein can activate a reporter containing a TA-rich
PLZF binding site, whereas PLZF-RAR has no effect on these genes.
This information suggests that the RAR-PLZF protein could act in a
dominant negative manner, binding and altering transcription of PLZF
target genes. By fusing portions of PLZF to the heterologous GAL4 DNA
binding domain, the PLZF protein was found to contain two separable
transcriptional repression domains, one of which overlaps the POZ/BTB
domain.394 Similarly, the POZ/BTB domains of the Bcl-6 and
ZF5 proteins were found to mediate transcriptional
repression.374-377 It is not certain whether the same
portions of the POZ/BTB motif are required for PLZF repression,
self-association, localization into subnuclear speckles,289,383 and association with cofactors. However,
we recently created missense mutations in the POZ/BTB domain that abrogated repression and that will aid in the molecular
characterization of the POZ/BTB domain.605 The mechanism of
transcriptional repression by PLZF is rapidly becoming elucidated. PLZF
was found to interact both in vitro and in vivo with the corepressors
N-CoR and SMRT and Sin3A and HDAC1 (histone deacetylase
1).293-295,299,397,398 These interactions occur via the
POZ/BTB domain of PLZF, although other regions of PLZF, including the
zinc finger motifs, may contibute to binding.296,397,398
This correlates with the fact that there are two repression domains in
PLZF, with one clearly mapping outside of the POZ
domain.394 PLZF, in turn, binds to specific regions of NCoR
and SMRT and sin3a and sin3b.295,296,397 SMRT colocalizes with PLZF in nuclear speckles and is able to potentiate the ability of
a GAL4-PLZF POZ/BTB domain fusion protein to repress
transcription.295 Transcriptional repression by PLZF was
potentiated by coexpression of the corepressors and only partially
blocked by the HDAC inhibitor trichostatin A,298 suggesting
additional mechanisms of repression other than alteration of
chromatin.399 Other investigators showed that the Bcl-6
POZ/BTB domain also associates with SMRT400 and other
members of the HDAC complex.397 Thus, it could be surmised that many POZ/BTB repressors work similarly by interacting with a
multiprotein repressor complex that contains N-CoR, SMRT, sin3A/b, and
histone deacetylases, leading to alterations of chromatin configuration.46 Additional mechanisms could be at play as
well, because our group found that PLZF forms a DNA-protein complex with a molecular weight of nearly 600 kD that contained
cdc2,401 which was implicated in transcriptional repression
by phosphorylation of basal transcription factors.402
| |
GROWTH SUPPRESSION BY PLZF |
PLZF is similar to PML167,184,210,247,248 in that both
proteins can repress cell growth. Pools of the interleukin-3
(IL-3)-dependent, nontumorigenic murine myeloid 32DCl3 cells
overexpressing the PLZF protein were highly growth inhibited when
cultured in IL-3, with their doubling time increasing from 18 hours to
greater than 3 days. These cells were retarded in the G1 phase of the
cell cycle and had a twofold to threefold increase in the spontaneous rate of apoptosis when grown in IL-3.403 Curiously,
PLZF-expressing cells also secreted a negative growth factor into
condition cell media that inhibited the growth of non-PLZF-expressing
cells. PLZF expression was also associated with inhibition of myeloid differentiation induced by G-CSF or GM-CSF, upregulation of the early
hematopoietic marker Sca1, and downregulation of the differentiated granulocytic marker Gr1. The molecular mechanism of action of PLZF on
cell growth is beginning to be elucidated. Acute infection of myeloid
cells with a PLZF-containing retrovirus was associated with growth
arrest of cells in the S-phase of the cell cycle. Progress of cells
from G1 into S phase is largely controlled by phosphorylation events
mediated by cyclin A paired with CDK2.404,405 Fibroblasts
expressing PLZF were growth suppressed and showed blunted induction of
cyclin A when stimulated from G0 into the cell cycle by
serum. PLZF can bind two sites derived from the cyclin A promoter and
can downregulate the cyclin A2 promoter in cotransfection
experiments.406 Furthermore, PLZF-expressing, growth-suppressed 32DCl3 cells regain a normal rate of cellular growth
when superinfected with a cyclin A-containing retrovirus. These data
suggest that cyclin A is a bonafide target gene of PLZF and that PLZF
can inhibit cellular growth in a variety of cell types by altering the
expression of regulators of the cell cycle.
PLZF expression was associated with protection of 32D
cells403 from apoptosis associated with IL-3 withdrawal,
suggesting that PLZF might reset the set point between cell life and
death, possibly by affecting the expression of bcl-2, bcl-x, bad, or other regulators of apoptosis.407 It is interesting to
speculate that high-level PLZF expression plays a role in the
quiescence and resistance to apoptosis exhibited by hematopoietic stem
cells. Downregulation of PLZF during myeloid differentiation may be
accompanied by cycles of committed cell division. It might be argued
that PLZF is actually a tumor suppressor disrupted in
t(11;17)(q23;q21)-APL. The resulting PLZF-RAR fusion proteins may
then act as a dominant negative inhibitors of normal PLZF function (see
below). Hence, t(11;17)(q23;q21) APL cells might be functionally null
for PLZF. Disruption of PLZF function may also play a role in t(15;17)
APL, because PML-RAR delocalizes PLZF in these cells and may thus interfere in its function.216,299
| |
PLZF-RAR |
The t(11;17)(q23;q21) fusion yields two reciprocal transcripts
(PLZF-RAR and RAR/PLZF)361,362 (Fig 6). The
breakpoint within the PLZF gene occurs 3' to the first translated
exon. As a result of the fusion, the PLZF-RAR chimera contains the
entire N-terminal transcriptional effector region of PLZF (including
the POZ/BTB domain) as well as the first two zinc fingers of the
protein. As in all forms of APL, the RAR gene is fused in the region
corresponding to the B domain. In one case, a fusion of the N-terminus
of PLZF and the first three PLZF zinc fingers (up to amino acid 484 of PLZF) was linked to RAR, indicating a breakpoint in the PLZF gene
further 3' within the gene. In 4 of 7 cases tested, a reciprocal RAR-PLZF transcript was detected, linking the A/AF1
ligand-independent transcriptional activation domain of
RAR42 to the last 7 zinc fingers of PLZF.
In comparing PML-RAR and PLZF-RAR, some interesting contrasts and
similarities can be defined. (1) Both fusion proteins can bind as
homodimers to RAREs.171,289,383 In the case of PML-RAR, this is mediated by the coiled-coil motif,171 whereas in
PLZF the POZ/BTB domain mediates self-association.383 When
coincubated with RXR, both PML-RAR and PLZF-RAR form multiple
different DNA-protein complexes. It is noteworthy that PLZF-RAR
homodimers bound to a direct repeat of the sequence GGG TCA separated
by 5 bp (DR5G) with equal avidity as PML-RAR but bound more strongly than PML-RAR to a repeat of the sequence GGT TCA
(Dr5T).383 This could potentially explain some of the
biological differences between PML-RAR- and PLZF-RAR-associated
APL. Although it is possible that PLZF-RAR homodimers might display
altered target gene specificity, in the presence of RXR, the
PLZF-RAR/RXR heterodimer binds to RAREs in vitro with higher
affinity than PLZF-RAR homodimers.289 However,
PLZF-RAR, produced by in vitro translation, bound less efficiently
to RAREs as a heteromer with RXR than the wild-type RAR.289 This may be due to POZ/BTB domain-mediated
multimerization, which could preclude efficient DNA binding (as seen in
other POZ/BTB proteins373), suggesting that PLZF-RAR
could form complexes that do not efficiently bind to DNA and that could
sequester limiting amounts of RXR, an essential cofactor for RAR function.
(2) Both PML-RAR13,15,157 and
PLZF-RAR289,383,408 can act in a dominant negative
manner to inhibit the activity of wild-type RAR and the vitamin D3
receptor (Perez et al171 and Licht and English, unpublished
data). PLZF-RAR is a relatively weak trans-activator, in some studies completely unable to activate transcription of coexpressed reporter genes408 and in our studies mediating
ligand-dependent transcription, albeit at levels less robust than the
wild-type RAR.289 The weakened transcriptional activity
of PLZF-RAR might be due to inefficient binding by the protein.
Alternatively, inclusion of the large PLZF moiety to the N-terminus of
the RAR might produce steric hindrance between RAR and
coactivators or basal factors. Deletion of the ligand-independent/AF1
activation domain of the RAR reduces activation by RAR, a
promoter-dependent effect.42 Most importantly, PLZF-RAR
interacts aberrantly with the SMRT and NCoR corepressors, Sin3A and
HDAC1, both in vitro and in vivo.293-296,299,397 A key
finding relates to the differential affinity of PLZF-RAR and
PML-RAR for NCoR and SMRT in the presence of ATRA. Whereas PML-RAR was able to release the corepressors and HDAC1 in the presence of 106 mol/L ATRA, PLZF-RAR retained
corepressors and HDAC1 even under these high ligand
concentrations294-296,299,409
(Fig 8).
PML-RAR association to HDAC1 and corepressors is mediated solely by
the corepressor binding domain or CoR box of the RAR moiety. In
contrast, PLZF-RAR binds to corepressors via the CoR box binds and
the POZ/BTB domain. It is the latter association that is insensitive to
ATRA even at high doses.295,296 This model is supported by several studies. (1) Mutation of the CoR box of PML-RAR but not PLZF-RAR results in loss of binding to HDAC1 and corepressors. (2)
Mutation of the PML-RAR CoR box abolishes its ability of this
protein to block differentiation, whereas the PLZF-RAR CoR box
mutant inhibits differentiation, even in the presence of
ATRA.296 (3) HDAC inhibitors, such as trichostatin A and
sodium butyrate, were able to convert PLZF-RAR into an
ATRA-responsive transcription factor, presumably by inactivating the
remaining corepressor complex bound to the POZ/BTB domain. These
inhibitors also allowed U937 cells transfected with PLZF-RAR to
differentiate in the presence of ATRA.295,296
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| Fig 9.
Functional domains of the NPM protein and structure of
the NPM-RAR and reciprocal RAR-NPM proteins generated in
t(5;17)-APL. A relatively short N-terminal portion of NPM containing
the oligomerization domain of the protein is linked to RAR. In the
index patient, a longer form of the fusion protein was identified,
containing an additional sequences of uncertain origin. The reciprocal
RAR-NPM protein could potentially interact with wild-type NPM and
interfere with NPM functions.
|
|
When coexpressed with wild-type RAR, PLZF-RAR inhibits reporter
gene trans-activation by the wild-type receptor.289,383,408 This dominant negative effect of PLZF-RAR was partially relieved by
overexpression of RXR, consistent with the notion that the aberrant
receptors block myeloid differentiation at least partly by limiting the
ability of RAR to bind with RXR to its targets.289 Deletion mapping of PLZF-RAR protein also showed that dominant negative activity was dependent on the presence of the POZ/BTB domain.383 This region is also required for
self-association of PLZF-RAR and for formation of multimers that
could sequester RXR. Curiously, inhibition of wild-type RAR function
was partially dependent on the presence of the first two PLZF zinc
fingers, which are present in the fusion protein and which are also the binding site for PML. When the POZ/BTB domain and first two PLZF zinc
fingers were deleted from the fusion protein, PLZF-RAR became an
efficient activator of ATRA-mediated transcription.
The dominant negative effect of PLZF-RAR also suggests that it may
work by sequestering RAR transcriptional coactivators such as TIF1
or CBP in an inactive conformation, drawing them from RAR target
genes. This provides another explanation for why RXR only partially
rescues the dominant negative effect of PLZF-RAR. However, the
hypothesis that PLZF-RAR binds to RAR corepressors,45,410,411 inappropriately repressing RAR
target genes in the absence of ligand is more likely. In keeping with this, PLZF-RAR, like PML-RAR, inhibited the activity of an
RARE-containing promoter in the absence of exogenous
ATRA.299 Finally, PLZF-RAR could, in theory, also affect
the function of wild-type PLZF. In fact, PLZF-RAR and PLZF can
preferentially heterodimerize over the formation of PLZF homomeric
complexes.383 Hence, high-level expression of PLZF-RAR
in t(11;17)(q23;q21) blasts might sequester PLZF from binding to its
natural target genes and/or bind to limiting quantities of PLZF
transcriptional cofactors.349 However, we have not yet
observed this effect in transfection experiments (Li et al, unpublished data).
| |
MODELS OF t(11;17)(q23;q21) APL |
Cellular models are being developed to clarify the role of PLZF-RAR
in APL. We found that PLZF-RAR was only transiently expressed in
nontumorigenic 32DCl3 cells after retroviral infection, suggesting a
highly toxic or growth suppressive effect (Shaknovich and Licht,
unpublished data). Pelicci's group found that
PLZF-RAR could be stably expressed in HL60, TF1, and U937 leukemic
cells and blocks their differentiation in response to a number of
chemical inducers.349 The transformed state of these cells
might have allowed them to escape the potent growth-suppressive effects
of PLZF-RAR. Consistent with the above results, PLZF-RAR, unlike PML-RAR, failed to increase the sensitivity of transduced cells to
ATRA-mediated differentiation. HL60 cells lacking wild-type RAR
transduced with PML-RAR showed increased expression of RAR target
genes, whereas PLZF-RAR-transduced cells did not. Furthermore, whereas reintroduction of wild-type RAR or PML-RAR into the mutant HL-60 cells fully restored the ability of the cells to differentiate, as measured by the expression of leukocyte integrins, PLZF-RAR was only partially able to induce their expression. This
information suggests that the differences in induction of endogenous
genes by PLZF-RAR and PML-RAR are important for the ATRA-resistant clinical phenotype of t(11;17)(q23;q21)-associated APL.
More physiologically relevant data indicate that PLZF-RAR on its own
may not fully account for the ATRA resistance of t(11;17)(q23;q21) APL.
Marrow progenitor cells infected with a PLZF-RAR retrovirus are able
to be serially passaged ex vivo and displayed a primitive hematopoietic
phenotype. Upon treatment of these cells with 107
mol/L ATRA, proliferation ceased and differentiation
ensued.336
The role of the PLZF-RAR protein in leukemogenesis was further
explored in a transgenic model using the cathepsin G
promoter.299 These mice developed a chronic myeloid
leukemia (CML)-like syndrome rather than APL. As in the
case of the PML-RAR model, disease developed after a preleukemic
phase, suggesting that secondary mutations are required for
transformation. Acute leukemia developed more rapidly in these mice
compared with the PML-RAR transgenics, suggesting that PLZF-RAR
may be more oncogenic. Unlike PML-RAR transgenic mice, the
PLZF-RAR mice did not achieve complete remission after ATRA
treatment at a 106 mol/L dose, although they did
show some evidence of myeloid differentiation. PLZF-RAR transgenic
mouse leukemia cells treated with ATRA readily differentiated ex vivo,
whereas in vivo, PLZF-RAR mice required a higher dose of ATRA than
PML-RAR mice to induce short remissions.299 Thus,
animals harboring PLZF-RAR were not absolutely insensitive to ATRA,
suggesting that PLZF-RAR does not completely block ATRA-induced differentiation. The relative insensitivity of the disease in vivo
correlates with the impaired ability of PLZF-RAR to transactivate in
vitro, likely due to binding of corepressors even in the presence of
ligand. This notion was further confirmed by the fact that the histone
deacetylase inhibitor TSA in combination with ATRA synergistically
inhibited growth and induced differentiation of PLZF-RAR harboring
mouse leukemic cells. Therefore, the clinical phenotype of
t(11;17)(q23;q21) APL might partly be due to the inability to achieve
and sustain sufficiently high levels of ATRA required to stimulate the
PLZF-RAR fusion product (Table
5). A combination of ATRA and sodium butyrate, the latter
already in clinical trial, might alleviate this
situation.412,413 These animal experiments are still
imperfect models of APL. PLZF-RAR mice, unlike PML-RAR mice, did
not accumulate promyelocytes,299,342 although transgenic
mice expressing PML-RAR under the cathepsin G promoter did not
develop true APL either.341,342 In addition, the animal
model does not explain the poor response of these patients to
chemotherapy and the resistance of fresh t(11;17)(q23;q21) APL cells to
high doses of ATRA in vitro.360,361,414 This information implicates another oncogenic lesion, potentially the reciprocal RAR-PLZF protein in the aggressive nature of this form of APL. This
notion is supported by recent studies with fresh APL blasts of a
patient with t(11;17)(q23;q21) APL.414 Neither ATRA nor arsenic treatment of these cells led to differentiation or apoptosis of
the APL cells. Although arsenic can eliminate the microspeckled pattern
of PML-RAR expression in APL associated with t(15;17), arsenic did
not affect the punctate nuclear appearance of PLZF-RAR. ATRA
treatment led to the degradation of PLZF-RAR, theoretically lifting
the block to induction of RAR target genes, yet did not induce
differentiation, suggesting that the reciprocal product offered a
second oncogenic lesion.
| |
RAR-PLZF |
In the case of the t(11;17)(q23;q21) APL variant, the reciprocal
transcript encoding RAR-PLZF that yields a protein containing the
last seven zinc fingers of PLZF fused to the A-domain of
RAR361,362 (Fig 6) is consistently expressed. In
contrast, the RAR-PML transcript of t(15;17) APL is absent in a
significant number of cases.5 These seven zinc fingers can
bind to the artificial PLZF binding site derived from PCR-based site
selection as well as a site derived from selection from a human CpG
island library.415 RAR-PLZF demonstrates properties that
may be critical to the disruption of transcriptional and nuclear
regulatory events. Whereas PLZF represses gene transcription through
its cognate binding site, RAR-PLZF activates transcription through
this site.394 Whereas PLZF is a growth suppressor and
inhibits expression of the cyclin A2 gene, RAR-PLZF activates
transcription, activates expression of cyclin A2 in an adhesion
independent manner in 3T3 cells,406 and enhances cell
growth (Yeyati et al, unpublished data). Hence, t(11;17)(q23;q21) may be an ATRA and chemotherapy-resistant disease due
to the presence of two oncogenes working through different mechanisms,
PLZF-RAR blocks retinoid-mediated activation of genes critical for
myeloid differentiation. RAR-PLZF may activate cell cycle regulators
such as cyclin A, accelerate cell growth, and block the
antiproliferative effects of retinoid treatment. This notion is
supported by the finding that mice harboring the RAR-PLZF protein
develop a myeloproliferative syndrome. Whether these mice or the
progeny of RAR-PLZF/PLZF-RAR crosses will develop leukemia is
under study.416
Finally, RAR-PLZF might also function through interference with
RAR-mediated signaling. Inappropriate expression of this fusion
protein, containing part of the AF1 activation domain of RAR, may
act competitively with wild-type RAR for limiting cofactors. Similarly, a truncated form of RAR containing the AF1 domain inhibited the ability of wild-type RAR to activate
transcription417 and transformion of keratinocytes,
blocking their ability to differentiate. This supports the idea that a
reciprocal RAR-X fusion protein could have an independent oncogenic effect.
| |
NUCLEOPHOSMIN (NPM-RAR) |
In t(5;17)-associated APL, RAR is translocated to a region on
chromosome 5q35 encoding the ubiquitously expressed and evolutionarily conserved nucleophosmin gene (NPM).418-420 NPM, also known
as B23, numatrin, and NO38, was initially isolated as a nucleolar
phosphoprotein in hepatoma cells.421-423 The human NPM gene
spans 25 kb, consists of 12 exons,424 and has a promoter
region consistent with those of housekeeping genes.425,426
Alternative splicing yields two major isoforms: NPMB23.1
(294 amino acids) and NPMB23.2 (257 amino acids), differing
in their C-terminal region.420,427-429 Major structural
features of NPM include two Asp/Glu-rich acidic domains, which may
serve as binding sites for basic regions of other proteins; a bipartite
nuclear localization signal (NLS); a metal binding motif; an ATP
binding site; phosphorylation sites for cdc2 kinase and casein kinase
II; and a binding site for proteins that contain nucleolar localization
signals420,430-440 (Fig 9). The C-terminal region of the
294 amino acid NPMB23.1 isoform is also involved in nucleic
acid binding and stimulation of DNA polymerase
activity.441-444 Both isoforms reversibly multimerize to a
hexameric state445 via the N-terminal
domain.446
NPM is localized most prominently to areas of the nucleolus
associated with ribonucleoprotein (RNP)
processing.235,237,447-449 NPM binds to nucleic
acids, altering their conformation, an effect that could facilitate
binding of ribosomal proteins to rRNA.237 NPM also
copurifies with proteins required for DNA
replication.443,450-452 In addition, NPM, particularly at
the N-terminal region, is highly homologous to the evolutionarily
conserved protein nucleoplasmin, a factor with chaperone activity that
is involved in chromatin/nucleosome assembly.453,454 NPM
also functions as part of a transport system used by ribosomal
precursors to shuttle between the cytoplasm and
nucleolus.236,440,455 NPM interacts with nonribosomal
proteins via basic sequences (NoLS), assisting in transport to the
nucleolus.436,455,456
NPM levels are increased in proliferating cells457,458 and
hypertrophic tissue to even higher levels in malignant
cells,420,459,460 including leukemic blasts.461
The increase in NPM expression may just be the consequence of increased
requirements for ribosomal precursors. However, engineered
overexpression of NPM in 3T3 cells yielded a transformed
phenotype.461 One explanation for this could be that NPM
binds to the tumor suppressor IRF-1 and inhibits its ability to
activate genes that mediate the antiproliferative effect of
IFN.461 Thus, NPM could behave as an antitumor suppressor.
NPM binds to transcription factor YY1, which is involved in cell growth
and differentiation, changing it from a transcriptional repressor to an
activator.462,463 YY1 binds to the NPM enhancer, possibly
constituting a feedback mechanism.428 ATRA-induced
differentiation of HL60 cells, but not growth arrest by serum
withdrawal, resulted in downregulation of NPM.464 When
cellular NPM levels were decreased by an antisense oligonucleotide,
there was potentiation of the ATRA-induced
differentiation.464 Growth-suppressive IRF-1 is upregulated
by ATRA during myeloid differentiation, in opposition to the effect of
NPM.115 These results support a possible role for NPM in
the control of cellular growth and differentiation and hint at
involvement in retinoid and IFN pathway regulatory cross-talk.
NPM undergoes dynamic changes over the course of the cell cycle.
Expression peaks at S or G2 phase and is minimal in cells at
G0.460,465-467 This pattern of expression might
be related to the fact that NPMB23.1 specifically
stimulates the activity of DNA polymerase .444 Alternatively, this may be a reflection of metabolic demands. In
addition, NPM undergoes cdc2 threonine phosphorylation in
G2/M.468,469 During M-phase progression, NPM associates
with perichromosal regions and prenucleolar bodies, thus functionally
linking the processes of nucleolar disassembly to mitotic chromosome
condensation.469 The fact that NPM is intimately involved
in events taking place at the G2/M regulatory point also underlines the
strong relation between this protein and cellular proliferation.
NPM is preferentially regulated during apoptosis and cell damage.
Apoptotic prostatic cells have hypophosphorylated NPM due to decreased
CK II activity. A protease specific for unphosphorylated NPM then
degrades the protein.470,471 NPM undergoes ADP-ribosylation after cells are exposed to x-rays, suggesting a role for NPM in DNA
repair.472 NPM reversibly delocalizes from the nucleolus to
the nucleoplasm235,473 when cells are exposed to conditions that discourage DNA or RNA synthesis or encourage terminal cell division, including stationary growth, serum starvation, hyperthermia, chemotherapeutic drugs, or ATRA (in HL-60
cells).235,473-483 This may reflect either decreased
shuttling due to diminished metabolic requirements or a controlling
role for NPM in regulating the cessation of cell growth. The dynamic
response of NPM after various cell stresses resembles the
reorganization of the PML-containing nuclear body under such
conditions, indicating that both proteins may measure or control cell
homeostatic processes.
NPM is fused to genes other then RAR in hematologic malignancies, as
in the t(2;5)(p23;q35) translocation found in Ki-1+
anaplastic large-cell lymphoma (ALCL).425,484-495 In this
situation, NPM is linked to ALK, a gene encoding a membrane spanning
tyrosine kinase425 normally not expressed in lymphoid
tissue.496 The resulting protein contains the N-terminal
oligomerization domain of NPM intracytoplasmic to the tyrosine kinase
domain of ALK.424,497 As a result, the ubquitously
expressed NPM gene drives the expression of an aberrant tryosine
kinase, which can multimerize, yielding constitutive kinase activity,
and aberrant oncogenic signaling.490,495,498-502 In the
t(3;5)(q25.1;q34) translocation, found in myelodysplasia and M6-AML, a
larger 175 amino acid portion of the NPM gene is linked to the MLF1
gene encoding an abundant cytoplasmic protein of unknown
function.424,503 The resulting NPM-MLF fusion aberrantly places the MLF moiety in the nucleus and nucleolus. This fusion protein
might also block normal NPM function, disrupting normal cellular growth
control mechanisms. A similar mechanism could be at play in the case of
NPM-RAR.
| |
NPM-RAR |
The t(5;17)(q35;q21) translocation was first described in a 2-year-old
girl with APL504 who achieved cytogenetic remission after
treatment with ATRA and chemotherapy. Blasts from this index patient,
when thawed and treated with ATRA, differentiated into mature
granulocytes.505 Subsequently, at least two other cases were described,418,506 one of whom has had a prolonged
survival after ATRA and bone marrow transplantation.384
Like the t(11;17)(q23;q21) APL syndrome, t(5;17) patients present with
atypical morphological features, but unlike PLZF-associated APL, these
patients are sensitive to ATRA therapy. This gene rearrangement joins
the NPM gene 5' to exon 3 of RAR in a manner similar to the
other forms of APL.418,505 From a cDNA library derived from
the marrow of the index patient, two fusion cDNAs were isolated. In the
NPMS-RAR (520 aa), NPM contributes its oligomerization,
metal binding, and nucleoplasmin homology domains as well as its highly
active promoter. NPML-RAR (563aa) harbors an additional
sequence of unclear origin, possibly from the noncoding region of NPM
(Fig 9).
Like PML-RAR and PLZF-RAR, the NPM-RAR fusion acts as a
ligand-dependent transcriptional activator when coexpressed with reporter genes containing RAREs,419 although it is not yet
known whether NPM-RAR can act as a dominant negative inhibitor of
wild-type RAR. This is likely, because the NPM moiety of the fusion,
like the PML-and PLZF proteins, contains a multimerization domain and hence could sequester RXR and other cofactors from wild-type RAR. NPM-RAR has biological effects in cell culture similar to PML-RAR and PLZF-RAR.349 Its engineered expression in U937 cells
blocked monocytic differentiation in response to vitamin D3
and TGF.507 This furthers the notion that all of the
RAR fusion proteins elicit the APL phenotype through disruption of
nuclear receptor signaling. This was supported by the finding that
PML-RAR, PLZF-RAR, and NPM-RAR could enhance the proliferation
of primitive marrow progenitor cells after retrovirus-mediated gene
transfer. Treatment of these cells with ATRA induced differentiation
and inhibited cell growth.336 A transgenic model of
t(5;17)-APL was recently created using the cathepsin G promoter. These
mice developed an APL-like syndrome after a latent period, and blasts
derived from these animals were ATRA sensitive.508 The
response of this disease to ATRA may be mediated in part by
downregulation of transcription of the NPM-RAR fusion, akin to the
decline of NPM levels in differentiating HL60 cells.464
This would relieve the putative dominant negative effect of the fusion protein.
NPM-RAR is expressed in a microspeckled pattern throughout the
nucleus, similar to PML-RAR and PLZF-RAR.384 In this
regard, NPM-RAR probably does not affect PML function, because PML
was expressed in the wild-type nuclear body configuration in HeLa cells
engineered to overexpress NPM-RAR, whereas NPM-RAR was expressed
in a diffuse nuclear pattern234 and APL blasts from a
t(5;17) patient exhibited a normal NB configuration.384
Furthermore, NPM did not interact with PML in vitro. Interestingly,
these experiments supply additional evidence that disruption of at
least the PML component of the nuclear body is not required for the
pathogenesis of APL.234 However, PLZF was delocalized
in t(5;17) APL cells in a microspeckled pattern distinct from its
wild-type distribution in normal marrow progenitor
cells.384 This finding further supports a broad role for
the PLZF protein in the pathogenesis of APL.
A reciprocal RAR-NPM mRNA was identified in the index t(5;17)
patient, leading to fusion of the A domain of RAR to the acidic domains, NLS, and the rest of the C-terminus of NPM419 (Fig
9). This molecule could associate with wild-type NPM through C-terminal sequences; however, it is unknown whether it interferes with any NPM
function such as nucleolar-cytoplasmatic shuttling or apoptosis. RAR-NPM may bring the RAR activation domain in contact with NPM
transcriptional targets such as YY1 and IRF-1, altering NPM-dependent transcriptional modulation. Lastly, similar to the other reciprocal fusion proteins, the ectopic expression of the RAR activation domain
could interfere with wild-type RAR function by competition for AF1 cofactors.
| |
NUCLEAR MATRIX-MITOTIC APPARATUS PROTEIN (NuMA) |
The gene encoding the NuMA protein is the newest reported fusion
partner of RAR in APL and the first mitotic apparatus protein found
to be genetically rearranged in a human malignancy.509 NuMA
is a large, highly abundant, conserved, and ubiquitously expressed
protein that is intimately involved in the completion of mitosis and
reformation of nuclei in the postmitotic daughter cells. NuMA is also a
structural unit of the interphase nucleus.510-515 NuMA was
first identified as an insoluble nonhistone chromatin-associated protein distributed diffusely throughout the interphase nucleus and
displays a remarkable crescent-shaped mitotic staining
pattern.516,517
The NuMA gene, located on chromosome 11q13,518 encodes a
highly conserved protein of 2,115 amino acids with a molecular weight of approximately 230 kD and by alternative splicing yields 1776aa and
1763aa proteins.519,520 The NuMA protein is divided into two globular domains at either end of the protein, with a central coiled region of 1485 aa515,521-523
(Fig 10). The coiled
motifs likely mediate protein homoassociation and
heteroassociation.521,524 Little is known about the
N-terminal globular region, although its absence impairs
nuclear reformation after mitosis.522,525 The C-terminal
region contains basic sequences, motifs for phosphorylation by cdc2 and
other kinases,521,526 and sequences that confer
localization to the nucleus (NLS) and mitotic
spindle.520-522,525,527,528
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| Fig 10.
Functional domains of the NuMA protein and
structure of the NuMA-RAR protein generated in t(11q13;17)-APL. As
in the other fusion proteins of APL, an oligomerization domain
contributed in this case from NuMA is linked to RAR. It is uncertain
if a reciprocal protein is generated.
|
|
There is experimental evidence to support a role for NuMA in mitosis,
apoptosis, and interphase nuclear matrix functions. It can participate
in these diverse processes due to complex regulatory posttranslational
modifications across the cell cycle. For example, NuMA is
phosphorylated by the cdc2/cyclin B-regulatory kinase at the initiation
of mitosis526,529 and associates with the spindle microtubules. A number of experiments demonstrated that NuMA is essential in forming convergent mitotic spindles and organizing and
stabilizing parallel arrays of
microtubules.510,511,525,527,530-535 NuMA may be required
for the proper organization of newly forming daughter nuclei as cell
division ends,511,517,527,531,532,536 forming a pathway for
proteins to transit to daughter nuclei.515 Towards the end
of mitosis, NuMA undergoes proteolytic cleavage and
dephosphorylation.526,529 Furthermore, as cells progress towards G1, the remaining NuMA reverts to an insoluble form, yielding a
fibrous network that may play a structural role during interphase. NuMA, like NPM, is an important target for regulation at the G2/M boundary as well as during postmitotic reorganization.
In interphase cells, immunofluorescence staining with anti-NuMA
antibodies shows diffuse and/or speckled nuclear
patterns.511,517,530,537 NuMA associates with a subset of
nuclear core filaments around which proteins and nucleic acids coalesce
to form the nuclear matrix.538 PML, NPM, and probably PLZF
associate with the nuclear matrix.191,197,216,460,467 Other
roles for NuMA were proposed based on coprecipitation with snRNPs and
association with splicing complexes.539 In addition, NuMA
specifically attaches to DNA matrix attachment regions (MAR), which are
important for chromatin compaction and isolation of transcriptionally
active loops of DNA.540,541 NuMA may thus provide
structural support for RNA processing pathways, organize chromatin
condensation, and participate in the regulation of transcriptional units.
NuMA may play a role in apoptosis and is an early target for
proteolysis by caspase-3 and caspase-6,542 yielding an
approximately 180-kD NuMA proteolytic product,526,543,544
lacking the C-terminal globular effector domain after several different
apoptotic stimuli.544-547 This proteolytic product could
act as a dominant negative, disrupting normal nuclear structure.
Alternatively, release of NuMA from the DNA matrix attachment regions
may facilitate the DNA fragmentation characteristic of
apoptosis.546 However, in some of these experimental models, there was no correlation between nuclear fragmentation and NuMA
proteolysis,544 suggesting that NuMA may be specifically targeted to prevent dying cells from proceeding through
M-phase. In summary, NuMA would appear to be a structural
component of the cell that responds to cell cycle signals on cue rather
than a controlling factor in cell proliferation. It is unclear whether inhibition of NuMA function might contribute to oncogenesis. In fact,
many NuMA mutants seriously disrupt mitosis and would not be expected
to be compatible with normal cell growth and proliferation.
| |
NuMA-RAR |
The NuMA-RAR fusion protein was first described in a 6-month-old boy
with morphologically diagnosed APL harboring a translocation t(11;17)(q13;q21).509 The patient had a complete remission
after ATRA therapy and was disease free at 24+ months after a bone
marrow transplant.509 The t(11;17)(q13;q21) results in a
2286 aa protein predicted to consist of 1883 amino acids of NuMA,
including the N-terminal globular and coiled-coil domains of NuMA fused
to RAR domains B through F, as in all the other RAR fusion
proteins548 (Fig 10). NuMA-RAR localized to sheetlike
nuclear aggregates in the patient's leukemic cells, but NB structure
and PML staining were unperturbed. Introduction of C-terminal deletion
mutants of NuMA into cells completely disrupts
mitosis,522,525 and it is surprising that the existence of
NuMA-RAR is even compatible with cell division. Perhaps the RAR
moiety mislocalizes the mutant NuMA from the daughter nuclei reforming
after mitosis, allowing the remaining wild-type NuMA to perform its
function. In the nucleus, the fusion may, in a dominant negative
manner, interfere with regulatory events during interphase or G2.
NuMA-RAR might also compete with wild-type NuMA for caspases,
interfering with apoptosis.
The most likely mechanism of action of the NuMA-RAR is interference
with nuclear receptor function. Although no functional data for the
NuMA-RAR fusion are yet available, it is probable that, like the
other RAR fusion proteins, NuMA-RAR is a dominant negative
retinoid receptor with diminished intrinsic transcriptional activity. A
fundamental common denominator among APL fusion proteins is their
ability to form higher order complexes. Like PML, PLZF, and NPM, NuMA
can multimerize.521,524 NuMA-RAR might thus sequester RAR partner proteins or have aberrant affinity for nuclear receptor coactivators or corepressors. Like PML, NuMA is a component of the
nuclear matrix and NuMA-RAR might further inhibit RAR function by
confinement of RAR cofactors in a subnuclear compartment (such as
the MAR) apart from wild-type RAR. Another common theme among RAR
fusion proteins is their downregulation when treated with ATRA.
Similarly, NuMA-RAR might decrease as t(11;17)(q13;q21) APL cells
are induced to terminally differentiate by ATRA. This is consistent
with the fact that normal neutrophils do not express NuMA.547 Finally, it is unknown whether a reciprocal
RAR-NuMA transcript is expressed in this disease. In fact, there
might be selection against this protein as it could represent a
detriment to mitosis.
| |
COMMON THEMES, SPECULATIONS, AND DEPARTURES FOR FUTURE INVESTIGATION |
There are three axes to be investigated in understanding the
pathogenesis of APL (Fig 11).
First, it is clear that certain RAR target genes must be activated
(or repressed) for myeloid development to proceed. In all APL patients,
one allele of RAR is disrupted and fused to the partner gene, N. All
of the N genes encode a protein able to multimerize. The
multimerization of the N-RAR fusion protein may be a key factor in
interfering with normal ATRA-mediated signaling and sequestering
cofactors. Recent evidence shows that PML-RAR and PLZF-RAR have
abnormally high affinities for corepressors. Because PML alone does not
seem to bind these factors, it appears that fusion of RAR to the PML
moiety effects an allosteric change in the entire molecule, causing the
fusion protein to retain the corepressors at physiological
concentrations of ATRA. The structural basis of this change is unknown.
NPM and NuMA might have the same effect on RAR, and this should be
investigated. In contrast, PLZF binds the corepressors even in the
presence of ATRA. The net result is that, at physiological levels of
ATRA, the fusion receptors do not activate the critical genes for
myeloid differentiation and only can do so with pharmacological doses.
In the case of PLZF-RAR, even the usual pharmacologic dose of ATRA
is insufficient for full activation of the RAR axis. The use of of
105 mol/L ATRA and/or addition of HDAC inhibitors
are required to allow retinoic acid signaling to proceed.
Second, the N gene is disrupted with only one normal allele remaining.
Loss of N gene dose and/or function might play a role in the
pathogenesis of APL. The N-RAR fusion could sequester the normal N
product, altering its role in growth control. PML-RAR delocalizes
both PML and PLZF from nuclear bodies, potentially altering their
function. Whether NPM-RAR and NuMA-RAR affect the function of the
wild-type proteins is unknown.
Third, the reciprocal RAR-N fusion gene could play a role in the
pathogenesis or clinical phenotype of the APL syndrome. The N-RAR
fusion, in addition to altering RAR function, could act to as a
second dominant negative protein to inhibit the function of the N
protein. In the cases of t(11;17)(q23;q21)-associated APL, the
RAR-PLZF gene product could both interfere with normal PLZF function
and have novel gains of function. Hence, many questions remain,
including the following:
(1) What are the critical RAR target genes required for myeloid
differentiation that are inhibited in their expression by the N-RAR protein?
(2) What are the molecular details of the corepressor complex bound to
N-RAR that inhibit the activity of the fusion protein? Are all the
corepressors directly bound or are a chain of protein-protein interactions required? Are coactivators also sequestered by the N-RAR fusions? Will manipulation of coactivators (histone
acetylases) as well as corepressor histone deacetylase factors play a
role in restoration of normal myeloid differentiation and the treatment of APL?
(3) Is the NB an active organelle or an intranuclear storage site? What
is significance of the PML and PLZF association with the NB? Is there a
role for NB disruption in the pathogenesis of APL?
(4) What is the mechanism of action of the PML protein in
transcription, apoptosis, and growth control?
(5) What is the role of PLZF in hematopoiesis and what are its target genes?
(6) How do RAR fusion proteins transform cells? Do the fusion
proteins alter expression of regulators of apoptosis? Are the RAR
fusion proteins dominant negative inhibitors of PML or PLZF? Will APL
develop both in PML or PLZF null animals?
(7) How does arsenic cause the apoptosis of APL cells? Is arsenic
response dependent on the presence of N-RAR fusion proteins or is it
reflective of the particular state of differentiation of the APL cell?
(8) Do RAR-N reciprocal fusion proteins cooperate with N-RAR
proteins to induce leukemia?
(9) Can other molecular pathways, such as caspases or IFN mediators, be
exploited as avenues for future therapies of APL?
(10) Are there molecular interactions between PML and NPM or NuMA?
The study of the molecular pathogenesis of APL is at the forefront of
the application of molecular biology to clinical medicine, because this
disease is the paradigm for successful differentiation therapy. The
spectacular response of these patients to ATRA has underlined the
importance of continued efforts to understand the basic biology of
leukemia. We now understand that the RAR fusion genes of APL are the
key to the cause and cure of this disease. Work to date has elucidated
how the RAR fusion proteins may block differentiation and how ATRA
can reverse this block and promote differentiation and death of the
malignant clone. Translational studies using clinical samples have
highlighted how retinoic acid resistance can occur in patients who
develop secondary mutations in the PML-RAR fusion gene. New work has
led to a greater understanding of how arsenic, a reactivated agent of
the cancer armamentarium, may promote death of neoplastic cells. The
occurrence of naturally resistant forms of APL such as that associated
with rearrangement of the PLZF gene indicates that molecular
heterogeneity can occur in APL and that a definitive diagnosis of
t(15;17)-APL must be made before ATRA can be used with confidence. The
study of the resistant and sensitive forms of APL, characterized by
rearrangement of the PML and PLZF genes, respectively, yielded an
appreciation of the importance of transcriptional repression by histone
deacetylation in the development of the disease. This led to the recent
use of the deacetylase inhibitor sodium butyrate as a form of targeted transcription therapy in a patient with resistant APL.549
With the development of animal and cell models of APL of sensitive and
resistant forms of APL and the advent of more powerful technologies for
gene discovery and cell biology, the next 5 years should offer continued insights leading to the development of more effective therapies for this fascinating disease as well as other forms of leukemia.
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ACKNOWLEDGMENT |
The authors thank S. Waxman, J. Gabrilove, G. Atweh, M. McConnell, and
A. Zelent for review of the manuscript and C. Brechok for editorial assistance.
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FOOTNOTES |
Submitted April 14, 1998; accepted December 29, 1998.
Supported by Grant No. R01 CA 59936 (J.D.L.) and American Cancer
Society Award DHP 160 (J.D.L.). J.D.L. is a scholar of the Leukemia
Society of America. A.M. is supported by Grant No. K08 CA73762.
Address reprint requests to Jonathan D. Licht, MD, Box 1130, Mount
Sinai School of Medicine, One Gustave L. Levy Place, New York, NY
10029; e-mail: jlicht{at}smtplink.mssm.edu.
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REFERENCES |
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INTRODUCTION
THE RETINOIC ACID RECEPTOR
AND MYELOID...
