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Blood, Vol. 93 No. 10 (May 15), 1999: pp. 3167-3215

REVIEW ARTICLE

Deconstructing a Disease: RAR&b.alpha;, 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.


    INTRODUCTION
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

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 alpha  (RARalpha ) gene to the promyelocytic leukemia (PML) gene on chromosome 15, yielding the fusion protein PML-RARalpha .10-15 These data suggested that disruption of RARalpha 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 RARalpha 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-RARalpha and RARalpha -N). This again highlights the importance of retinoid metabolism, but also suggests that partner genes with RARalpha could also play important roles. In this review, we will deconstruct the APL problem by evaluating the role of RARalpha in normal and neoplastic myeloid development. We will examine each of the genes fused to the RARalpha 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 RARalpha 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 RARalpha , 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 RARalpha is unique among these forms of APL in its resistance to differentiation therapy with ATRA or conventional chemotherapy.



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Fig 2. Functional domains of the RARalpha protein.


    THE RETINOIC ACID RECEPTOR
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

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), RARalpha is identified with myeloid development.27-29


    TRANSCRIPTIONAL FUNCTION OF RARalpha
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

RARalpha 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, RARalpha binds to retinoic acid response elements (RARE) located in the promoters of many genes, including those of RARalpha ,31,32 RARbeta ,33,34 and RARgamma .35 RAREs consist of a direct repeat (A/G)G(G/T)TCA separated by 2 or 5 nucleotides. RARalpha 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 RARalpha .39,40 RARalpha is a ligand-dependent transcription factor stimulated by ATRA, whereas its partner, RXR, responds to ATRA or 9-cis retinoic acid.41 RARalpha 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 RARalpha protein can have two different A domains (A1 or A2). The C-terminal E domain of RARalpha 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 RXRalpha and RARgamma 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 RARalpha 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 RARalpha 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 RARalpha 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.


    RARalpha AND MYELOID DIFFERENTIATION
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

The importance of RARalpha 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 RARalpha 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 RARalpha , RARbeta , or RARgamma .77,80 Furthermore, RXRalpha 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.

RARalpha may help program normal hematopoietic development. Erythroid induction of multipotent FDCP mixA4 cells by erythropoietin was correlated with complete downregulation of RARalpha expression, whereas myeloid differentiation induced by granulocyte colony-stimulating factor (G-CSF) was correlated with upregulation of RARalpha , particularly the RARalpha 2 isoform.81 Introduction of an RARalpha 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 RARalpha 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 RARalpha gene is only disrupted by the formation of chromsomal translocations yielding fusion genes (see below). The notion that the dominant negative RARalpha functions by sequestration of RXR into inactive complexes was supported by the finding that overexpression of wild-type RARalpha in murine bone marrow cultures85 led to the accumulation of promyelocytic colonies. Upon addition of ATRA, the RARalpha -expressing marrow colonies consisted mainly of more differentiated granulocytes. Hence, overexpression of wild-type RARalpha , C-terminal truncated forms of RARalpha and fusion proteins consisting of partners fused to the N-terminus of RARalpha (eg, PML-RARalpha ; 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 (10-7 to 10-6 mol/L) can overcome this block. How might this blockade occur at the molecular level? The fact that wild-type RARalpha as well as mutant forms of RARalpha 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 RARalpha and the basal transcriptional machinery. In support of this notion, in vivo footprinting of the RARbeta promoter shows occupancy of the RARE only under pharmacological ATRA concentrations.87 It might be predicted that forced expression of RXR and/or RARalpha coactivators would rescue the block by dominant negative RARalpha and allow differentiation to proceed at physiological ATRA concentrations.


    RARalpha TARGET GENES
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

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

                              
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Table 1. Genes Potentially Regulated by RARalpha in Myeloid Differentiation

Many of the target genes of RARalpha 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 RARalpha , correlating with the presence of a RARE in the second promoter of the RARalpha 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-RARalpha protein. This hypothesis is supported by recent data that indicate that PML-RARalpha 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 RARalpha , ATRA fails to induce the hoxb1,38,114 whereas RARgamma null cells fail to express hoxa1. This information suggests that in APL the disruption of RARalpha 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 (gamma -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 STAT1alpha at the mRNA level and increases tyrosine phosphorylation of STAT1alpha , together leading to a large increase in DNA binding activity of the STAT1alpha complex to an IFN-responsive element (IRE).120 RARalpha and STAT1alpha synergized to stimulate transcription from an IRE-containing reporter plasmid, whereas PML-RARalpha 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-epsilon , a newly identified basic-zipper transcription factor that recognizes CCAAT DNA sequences, was found to be rapidly upregulated during ATRA-mediated differentiation. C/EBP-epsilon 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-RARalpha downregulate C/EBP-epsilon expression in the absence of retinoic acid; the C/EBP-epsilon gene is then upregulated when pharmacological doses of ATRA are added to the cell.126 Hence, C/EBP-epsilon may be a model target gene of the PML-RARalpha 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 RARalpha in combination with RXR binds to an imperfect RARE within the human p21 promoter and RARalpha can activate the p21 promoter in a ligand-dependent manner. Therefore, p21 meets criteria for a bona fide RARalpha 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 RARalpha 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 RARalpha 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 RARalpha .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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Table 2. Evidence for Involvement of RARalpha in Myeloid Development


    PML
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
TRANSCRIPTIONAL FUNCTION OF...
RARalpha AND MYELOID...
RARalpha TARGET GENES
PML
NUCLEAR BODIES AND PML...
PML AND TRANSCRIPTION
PML PARTNER PROTEINS
PML, IFN, AND VIRAL...
PML, GROWTH SUPPRESSION, AND...
PML-RARalpha
TRANSCRIPTIONAL ACTIVITY OF THE...
PML-RARalpha AND RETINOID...
PML-RARalpha AND THE NUCLEAR...
CELLULAR MODELS OF PML-RARalpha ...
ANIMAL MODELS OF PML-RARalpha ...
SUGGESTED MODEL OF PML-RARalpha ...
RARalpha -PML
PLZF
PLZF NUCLEAR LOCALIZATION
PLZF EXPRESSION
TRANSCRIPTIONAL FUNCTION OF...
GROWTH SUPPRESSION BY PLZF
PLZF-RARalpha
MODELS OF t(11;17)(q23;q21) APL
RARalpha -PLZF
NUCLEOPHOSMIN (NPM-RARalpha )
NPM-RARalpha
NUCLEAR MATRIX-MITOTIC...
NuMA-RARalpha
COMMON THEMES, SPECULATIONS,...
REFERENCES

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-RARalpha and reciprocal RARalpha -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-RARalpha depicted, as well as the rarer intermediate form (not shown). The RARalpha -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).

(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-RARalpha 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-RARalpha 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.