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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3865-3878
Deregulated PAX-5 Transcription From a Translocated
IgH Promoter in Marginal Zone Lymphoma
By
Aline M. Morrison,
Ulrich Jäger,
Andreas Chott,
Michael Schebesta,
Oskar A. Haas, and
Meinrad Busslinger
From the Research Institute of Molecular Pathology, Vienna, Austria;
the University of Vienna Medical School, Vienna, Austria; and St. Anna
Children's Hospital, Vienna, Austria.
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ABSTRACT |
The PAX-5 gene codes for the transcription factor BSAP,
which is expressed throughout B-cell development. Although
loss-of-function mutation in the mouse showed an essential role for
Pax-5 in early B lymphopoiesis, gain-of-function mutations have
implicated the human PAX-5 gene in the control of late B-cell
differentiation. PAX-5 (on 9p13) has been involved together
with the immunoglobulin heavy-chain (IgH) gene (on 14q32) in
the recurring t(9;14)(p13;q32) translocation that is characteristic of
small lymphocytic lymphoma with plasmacytoid differentiation. Here we
have characterized a complex t(2;9;14)(p12;p13;q32) translocation
present in a closely related non-Hodgkin's lymphoma referred to as
splenic marginal zone lymphoma (MZL). In this MZL-1 translocation, the
two promoters of PAX-5 were replaced on the derivative
chromosome 14 by an immunoglobulin switch Sµ promoter that was linked
to the structural PAX-5 gene upstream of its translation
initiation codon in exon 1B. Expression analyses confirmed that
PAX-5 transcription was upregulated due to efficient initiation
at the Sµ promoter in the malignant B lymphocytes of patient MZL-1.
For comparison we have analyzed PAX-5 expression in another
B-cell lymphoma, KIS-1, indicating that transcription from the distal
PAX-5 promoter was increased in this tumor in agreement with
the previously characterized translocation of the immunoglobulin Eµ
enhancer adjacent to PAX-5 exon 1A. In both lymphomas, the
J-chain gene, which is thought to be under negative control by BSAP,
was not expressed, whereas transcription of the putative target gene
p53 was unaffected by PAX-5 overexpression. Together
these data indicate that the t(9;14)(p13;q32) translocation contributes
to lymphoma formation as a regulatory mutation that leads to increased
PAX-5 expression in late B-cell differentiation due to promoter
replacement or enhancer insertion.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
TUMORS OF HEMATOPOIETIC origin are
frequently associated with specific chromosomal translocations that
result in the activation of proto-oncogenes controlling
differentiation, proliferation, or cell survival. In B-cell
non-Hodgkin's lymphoma (NHL), these proto-oncogenes are often
deregulated by translocation adjacent to regulatory elements of
immunoglobulin genes. Most frequently, the immunoglobulin heavy-chain
(IgH) gene on chromosome 14q32 is involved in NHL that
encompasses a large spectrum of diseases with diverse morphological and
clinical manifestations. Different subtypes of NHL have been correlated
with characteristic chromosomal translocations and thus with the
activation of specific oncogenes. For instance, the c-MYC gene
on 8q24 is translocated in Burkitt's lymphoma, the BCL-1
(PRAD1) gene coding for cyclin D1 (on 11q13) is involved in
mantle cell lymphoma, and the antiapoptotic BCL-2 gene on 18q21
is activated in follicular lymphoma. In addition, the BCL-6
gene coding for a zinc finger transcription factor is frequently
altered by rearrangement in diffuse large-cell lymphoma.1-3
The t(9;14)(p13;q32) translocation has been closely associated with a
relatively rare subtype of NHL that was initially referred to as small
lymphocytic lymphoma (SLL) with plasmacytoid
differentiation.4 To date, this entity is listed under
B-SLL in the revised REAL classification of
lymphomas.5,6 This subtype of NHL is thought to originate
from peripheral B lymphocytes that have been stimulated to undergo
plasma cell differentiation.4,5 Consequently, this form of
B-SLL is characterized by the expression of cytoplasmic and
cell-surface immunoglobulin, by immunoglobulin secretion, and by an
indolent clinical course that may progress with time towards a more
aggressive large-cell lymphoma.4,5 The t(9;14)(p13;q32) translocation was first reported in the diffuse large-cell lymphoma KIS-1.7 The molecular characterization of this
translocation showed that the IgH locus was translocated in a
head-to-head configuration to the PAX-5 gene on chromosome
9p13. As a result, the potent Eµ enhancer of the IgH gene was
brought into close proximity to the PAX-5
promoters.8 In addition, this analysis showed that transcription of the PAX-5 gene is initiated from two promoters and thus results in splicing of two alternative 5 exons (1A or 1B) to common coding sequences (exons 2-10).8 Molecular
analysis of a second t(9;14) translocation identified breakpoints in
the switch Sµ region of the IgH locus and in the downstream
exon 1B of PAX-5.9 Moreover, a consistent
involvement of the PAX-5 locus in t(9;14) translocations of
B-SLL was confirmed by fluorescence in situ hybridization (FISH)
analyses using a 1 Mb-long yeast artificial chromosome probe spanning
the entire PAX-5 locus.9
PAX-5 codes for the transcription factor BSAP, which recognizes
its target genes via the highly conserved paired domain.10 Targeted inactivation of Pax-5 in the mouse germline showed an important role for this gene in B-cell and midbrain
development.11 During B lymphopoiesis, the Pax-5
gene is expressed from the earliest B-lineage-committed precursor cell
up to the mature B-cell stage and is subsequently downregulated during
terminal plasma cell differentiation.12,13 Consistent with
this expression pattern, Pax-5 is required for progression of
B-cell development beyond an early progenitor cell stage.14
Although several genuine BSAP (Pax-5) target genes have been identified
at the pro-B-cell stage by genetic means,15 little is
known about the function of Pax-5 in late B-cell differentiation. At
the mature B-cell stage, BSAP (Pax-5) has been implicated in the
control of cell proliferation and in the regulation of the J-chain
gene, the IgH germline  promoter, and the 3 enhancers
of the IgH and Ig loci.10 Because gain-of-function mutations are likely to provide new insight into the
role of Pax-5 in late B-cell differentiation, we were interested in
studying the effect of t(9;14)(p13;q32) translocations on the expression of PAX-5 and its target genes in human NHL.
Here, we describe the molecular characterization of a complex
t(2;9;14)(p12;p13;q32) translocation that was identified in a patient
diagnosed with marginal-zone lymphoma (MZL), a disease closely related
to B-SLL. This translocation generated a novel transcription unit by
linking a switch-Sµ promoter of the IgH locus (14q32) to the
structural PAX-5 gene in exon 1B (9p13). Efficient
transcription initiation from the Sµ promoter was directly shown in
the malignant B lymphocytes that constituted the majority of
mononuclear blood cells (MNC) in patient MZL-1. In contrast, PAX-5 transcription was increased from the upstream exon 1A
promoter in the KIS-1 lymphoma, thus reflecting the close proximity of this promoter to the translocated Eµ enhancer in these cells. Hence,
deregulation of PAX-5 expression by t(9;14) translocations can
be brought about by either promoter replacement or enhancer insertion.
The possibility is discussed that the expression of the PAX-5
gene under the control of IgH regulatory elements contributes to lymphomagenesis by interfering with the inactivation of
PAX-5 transcription and thus with terminal plasma cell
differentiation.
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MATERIALS AND METHODS |
Clinical data.
An 83-year-old woman presented with anemia, marked leukocytosis, and
splenomegaly at the Hematology Department of the Vienna Medical School
(Vienna, Austria) in December 1995. The white blood cell count was 31 × 109/L with 63% atypical lymphocytes that were
CD19+, IgM+, +, and displayed
low CD5 expression (Fig 1A). The serum did
not contain clonal IgM paraprotein. Bone marrow histology showed
multifocal lymphoid infiltrates within an otherwise normal parenchyma.
These infiltrates accounted for ~40% of the total bone marrow cells and were predominantly concentrated in the central part of the marrow
spaces. The infiltrating cells were frequently located in clusters
within the sinusoids of the bone marrow
(Fig 2A,B), which were recently described
as a characteristic hallmark of splenic marginal zone lymphoma
(MZL).16 The malignant cells in the blood were
characterized as medium-sized or moderately enlarged B lymphocytes with
round or slightly indented nuclei, clear cytoplasm, and frequent
cytoplasmic projections or thin villi (Fig 2D). Based on all these
data, the B-CLL tumor of this patient was diagnosed as splenic MZL in
agreement with the REAL classification.5,6 This patient,
referred to as MZL-1, was in excellent clinical condition and received
only supportive treatment with erythropoietin until September 1996, when thrombocytopenia and B-cell symptoms required chemotherapeutic
treatment. The patient received three courses of oral therapy with
chlorambucil (20 mg on day 1) and prednisone (50 mg on days 1 to 5).
After this treatment, the blood-cell count improved dramatically,
because the patient had a normal leukocyte count with 0% to 10%
atypical lymphocytes in monthly controls until July 1997. All analyses
described herein were performed with MNC of patient MZL-1 before
chemotherapy. Informed consent for the scientific use of these cells
was obtained from the patient.
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| Fig 1.
The mononuclear blood cells of patient MZL-1 consist
predominantly of malignant B lymphocytes. (A) Flow cytometric analysis
of peripheral blood cells of patient MZL-1. Mononuclear cells from the
blood of patient MZL-1 were analyzed by flow cytometry using a
FITC-conjugated anti-CD19 (HD37) antibody in combination with a
PE-labeled anti-CD5 (DK23) antibody or with unlabeled anti-IgM (AF6),
anti- (6E1), anti-CD23 (9P25), anti-CD38 (HB-7), and anti-CD40
(MoAb89) antibodies. The unlabeled MoAbs were visualized by indirect
staining with a secondary PE-conjugated goat antimouse antibody. (B)
Flow cytometric analysis of peripheral mononuclear cells of a normal
individual. The percentage of CD40+ B lymphocytes is
indicated.
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| Fig 2.
Morphological characterization of the malignant
B lymphocytes in the bone marrow and blood of patient MZL-1. (A,B)
Immunohistochemical analysis of bone marrow biopsies. Sections were
stained with anti-CD79a [Ig- ] (A) and anti-DBA.44 (B) antibodies.
Both antibodies stained lymphoid cells that were predominantly located
within the sinusoids of the bone marrow. The sinusoids are indicated by
arrowheads (cross-section) or arrows (oblique [A] and longitudinal
[B] sections), respectively. The clear spheroidal areas are occupied
by fat cells. (C) Staining of CD5+CD19+
peripheral blood cells. CD5+CD19+ cells
were sorted from mononuclear cells of the peripheral blood and stained
with the anti-DBA.44 antibody.25 About 70% of all sorted
cells were strongly positive for the DBA.44 antigen. (D) Morphology of
the malignant B lymphocytes. Blood smears of patient MZL-1 were
analyzed by the modified Wright staining. Four representative B cells
are shown at high magnification together with adjacent erythrocytes.
These B cells contain a round or ovoid nucleus that is surrounded by a
moderately increased amount of basophilic cytoplasm. Most malignant B
cells have the appearance of villous lymphocytes as evidenced by the
presence of cytoplasmic projections or short thin villi, which are
often asymmetrically located on one side of the cell.
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Immunohistochemistry.
The formalin-fixed, decalcified bone marrow trephine biopsy was
paraffin-embedded, sectioned, and stained with hematoxylin and eosin.
For immunohistochemistry, the sections were pretreated by microwaving
in citrate buffer (10 mmol/L, pH 6.0) twice for 5 minutes at 600 W
each. The sections were incubated with the monoclonal antibodies
(MoAbs) DBA.44 (DAKO, Glostrup, Denmark; 1:10) and CD79a (DAKO; 1:25)
for 1 hour followed by biotinylated horse antimouse immunoglobulin as
the secondary antibody and by Vectorstain Elite reagent (Vector Labs,
Burlingame, CA) and 3-amino-9-ethyl-carbazole (for DBA.44) or
3,3'-diamino-benzidine (for CD79a) as chromogens in the presence of
H2O2.
Frozen mononuclear cells of patient MZL-1 were thawed and sorted for
CD5+ CD19+ cells on a FACS Vantage TSO flow
cytometer (Becton Dickinson). Cytospin preparations of the sorted
CD5+ CD19+ cells were fixed with acetone on
glass slides for 10 minutes. The cells were incubated with the
anti-DBA.44 antibody for 1 hour, followed by incubation with
biotinylated horse antimouse immunoglobulin as a secondary antibody,
and then by alkaline phosphatase-conjugated streptavidin (Super
Sensitive HRP Label, Biogenex, San Ramon, CA). The reaction product was
visualized by new-fuchsin (DAKO) as a chromogen.
Cytogenetic analysis.
Bone marrow cells of patient MZL-1 were cultured and stimulated as
previously described.17 Metaphase chromosomes were prepared and G-banded with Trypsin/Giemsa according to standard techniques and
karyotyped with a PSI image analysis system (PSI, Halladale, UK). All
20 metaphase nuclei analyzed showed the same pseudodiploid karyotype:
46, XX, dup(1)(q12q32) add(1)(?p36), t(2;9;14)(p12;p13;q32), del(7)(q22q33), der(17)t(12;17)(q21;p12).
Cell lines.
The origin and culture conditions for the human B-cell lines BJA-B,
Raji, Namalwa, Ramos, and HS-Sultan, the myeloma cell line RPMI 8226, and the cervical carcinoma cell line HeLa have previously been
described.12,18 The human diffuse large-cell lymphoma line
KIS-119 and the myeloma cell line U226 (obtained from the
German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 1 mmol/L glutamine (GIBCO BRL).
Antibodies and flow cytometric analysis.
MoAb directed against the following human cell surface markers were
used for flow cytometric analysis. The FITC-conjugated anti-CD19 (HD37)
and PE-conjugated anti-CD5 (DK23) antibodies were purchased from DAKO,
the anti-CD38 (HB-7) MoAbs from Becton Dickinson (San Jose, CA), and
the anti-CD23 (9P25), anti-CD40 (MoAb89), anti-IgM (AF6) and anti-Ig
(6E1) from Immunotech (Marseille, France). A PE-conjugated
F(ab)2 goat antimouse IgG antibody (Immunotech) was used for
indirect staining of the unlabeled MoAbs.
Mononuclear cells were prepared from the peripheral blood of patient
MZL-1 and of a normal control individual by purification on Ficoll
gradients. Single-cell suspensions were incubated with the unlabeled
MoAb, washed, stained with the PE-conjugated F(ab')2 goat antimouse
IgG antibody, washed, and incubated with the FITC-conjugated anti-CD19
(HD37) antibody followed by analysis in a FACScan flow cytometer
(Becton Dickinson), as previously described.11
Preparation of genomic DNA.
DNA was isolated from ~107 cells of cultured human cell
lines, from mononuclear cells of peripheral blood, or from frozen lymph node biopsies that were ground into a powder in a mortar under liquid
nitrogen. These cell pellets were lysed and digested overnight at
37°C in 500 µL of a buffer containing 150 mmol/L NaCl, 50 mmol/L Tris pH 8.0, 1 mmol/L EDTA, 1% sodium dodecyl sulfate (SDS), and 2 mg/mL of proteinase K. Genomic DNA was subsequently isolated by
phenol-chloroform extraction and ethanol precipitation.
Southern blot analysis.
Restriction digestions were performed overnight with 10 µg of genomic
DNA at 37°C in a volume of 400 µL followed by ethanol precipitation and agarose gel electrophoresis. The separated DNA was
blotted onto Gene-Screen Plus membranes (DuPont, Boston, MA) by the
alkaline transfer method. DNA probes were radiolabeled by random
priming and hybridized overnight to the immobilized DNA at 65°C in
Church buffer (0.5 mol/L sodium phosphate pH 7.2, 7% SDS, 1 mmol/L
EDTA pH 8.0). Posthybridization washes were performed at 65°C for 3 × 20 minutes in 40 mmol/L sodium phosphate pH 7.2, 1% SDS. The
same membranes were rehybridized with different probes after stripping
in 0.1 mol/L NaOH/0.1% SDS for 20 minutes and neutralization in 0.5 mol/L Tris pH 8, 0.1% SDS for 15 minutes.
The 1.6-kb SacI-BstEII DNA fragment of clone
p8.5-17 was used as the exon 1A-specific PAX-5
probe #1 (pKIS). The exon 1B-specific probe #2 consisted of the 1.8-kb
PstI DNA insert of clone pD10-P1.8,8 whereas the
intron 2-specific probe #3 corresponded to a 1.23-kb SacI-HindIII DNA fragment of clone
pD10-N2.3.8
Cloning of translocation breakpoints.
HindIII-digested DNA prepared from MNC of patient MZL-1 was
fractioned on a 0.9% low-melting agarose gel, and the DNA fragments of
8 to 9 kb size were excised and purified. The size-fractionated HindIII fragments were ligated into the HindIII site of
the  ZAP Express vector (Stratagene) and packaged into
phage by the use of the Gigapack III Gold packaging extracts
(Stratagene). A library of 2 4 × 10 6 recombinant phages was separately screened with the
32P-labeled exon 1B-specific probe #2 and the intron
2-specific probe #3 for phages carrying the two reciprocal
translocation events. After plaque purification, the 8.6-kb DNA insert
corresponding with the translocation breakpoint on chromosome der(9)
was excised in vivo with the f1 helper phage R408 (Stratagene, La
Jolla, CA) from the ZAP Express vector into plasmid pBK-CMV to
obtain clone pTBP-der(9). The 8.9 kb HindIII fragment carrying
the reciprocal translocation breakpoint was recloned from phage DNA
into the pBluescript II KS(+) vector to generate clone pTBP-der(14).
The DNA sequence of the cloned breakpoints was determined on an
automated sequencer (PE Applied Biosystems, Foster City, CA) by primer
walking.
Riboprobes and RNase protection assay.
The following oligonucleotide pairs were used for PCR amplification of
the indicated human riboprobes:
The indicated oligonucleotides and total RNA of the human
B-cell line BJA-B were used to generate the following probes by RT-PCR
amplification and cDNA insertion into the polylinker of pSP64 in
antisense orientation relative to SP6 transcription: p53 (321 bp), Ex1A
(348 bp), J-chain (220 bp), and S16 (109 bp). The probe Ex2-4 of
PAX-5 was previously described.20 The probe Ex1B
was obtained by inserting a 915-bp Eag I-Pst I
fragment of clone pD10-P1.8 into the polylinker of pSP64.8
A 440-bp Sau3A-Pst I fragment spanning the IgH
Sµ-PAX5 exon 1B breakpoint was subcloned from clone
pTBP-der(14) into pSP64 to obtain the Sµ-Ex1B riboprobe.
Total RNA was prepared from human mononuclear cells and B-cell lines
using the TRIzol reagent (GIBCO BRL), and 10 µg of each RNA
preparation was used for RNase protection assay according to Vitelli et
al.21 The hybridization temperature was 60°C for all
riboprobes except for the Sµ/Ex1B and Ex1B probes that were hybridized at 70°C.
S1 nuclease analysis.
An S1 DNA probe containing the 5 end of PAX-5 exon 1B
was obtained by 5 end labeling the EagI end of a 563-bp
ApaI-EagI DNA fragment isolated from clone
pD10-P1.8.8 The S1 nuclease protection analysis was
performed as previously described22 except that the RNA-DNA
hybridization was performed at 60°C. A DNA sequencing ladder was
generated by depurination of the labeled DNA probe at G- and A-residues
as described.23
Electrophoretic mobility shift assay (EMSA) analysis.
Nuclear extracts were prepared from mononuclear cells of patient MZL-1
and from the cell lines KIS-1, BJA-B, and Raji as
described.24 Nuclear proteins (1 µg) were analyzed by
EMSA with an end-labeled oligonucleotide containing the paired
domain-binding site of the H2A-2.2 gene as described.12
Nucleotide sequence accession numbers.
The DNA sequences of the entire PAX-5 exon 1B and of the
translocation breakpoints on the derivative chromosomes 9 and 14 have
been submitted to GenBank (accession numbers AF074913-AF074915).
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RESULTS |
Characterization of the lymphoma cells of patient MZL-1.
Because the specific t(9;14)(p13;q32) translocation involving the
PAX-5 locus was highly correlated with SLL of the plasmacytoid subtype,4,8,9 we isolated DNA from affected lymph nodes of
18 B-SLL patients for Southern blot analysis with DNA probes specific
for the 5 region of the PAX-5 gene (see below). In
addition, DNA prepared from the peripheral blood of two patients
diagnosed with MZL and of one patient with prolymphocytic leukemia
(PLL) were included in the Southern blot analysis. Of all these
patients, an aberrantly migrating DNA fragment indicative of a possible translocation was detected in only one patient, here referred to as
MZL-1 (see below). In agreement with this observation, peripheral B
lymphocytes of this patient were shown, by cytogenetic analysis, to
contain a complex translocation involving 9p13 (PAX-5; see below). Patient MZL-1 initially presented with anemia, leukocytosis, and splenomegaly. In studying the malignant B lymphocytes of patient MZL-1, we characterized the mononuclear cells from the peripheral blood
by flow cytometry (Fig 1) and analyzed bone marrow biopsies by immunohistochemistry (Fig 2). Although a normal control individual contains only ~4% of CD40+ B cells in the MNC fraction
(Fig 1B), the blood of patient MZL-1 was almost exclusively composed of
malignant B cells (up to 94%; Fig 1A). These activated B lymphocytes
were weakly positive for CD5 expression (CD5low) and were
monoclonal with regard to the expression of a B-cell receptor
consisting of IgM and Ig. In addition, these cells were strongly
positive for CD19, CD23, CD40, and CD72, whereas they were negative for
CD3, CD10, CD21, CD28, CD38, CD43, CD56, Ig, IgA, and IgG expression
(Fig 1A; data not shown).
Analysis of bone marrow biopsies of patient MZL-1 showed massive
infiltrates of B lymphocytes that accounted for ~40% of all bone
marrow cells (Fig 2A and 2B), whereas only a few scattered mature B
cells are usually detected in normal bone marrow (data not shown). The
malignant B lymphocytes of patient MZL-1 were predominantly clustered
and aligned within the sinusoids of the bone marrow (Fig 2A and 2B),
which is considered to be a characteristic hallmark of lymphoma of
splenic origin.16 The infiltrating B lymphocytes were
reactive with antibodies directed against the B-cell marker CD79a
(Ig-; Fig 2A) and the antigen DBA.44 (Fig 2B). The DBA.44 antigen
was also detected on sorted CD19+ CD5+ B
lymphocytes from the peripheral blood of patient MZL-1, suggesting that
these peripheral B lymphocytes and the infiltrating B cells in the bone
marrow are of the same orgin (Fig 2C). The DBA.44 antigen is normally
found only on a subset of B lymphocytes in the mantle and marginal
zones of lymphoid follicles.25 Interestingly, the leukemic
B cells of patient MZL-1 resembled villous lymphocytes as evidenced by
the presence of cytoplasmic projections and fine villi (Fig 2D). The
characteristic morphology, bone marrow infiltration pattern, and
cell-surface phenotype of the malignant B lymphocytes therefore support
the diagnosis of splenic marginal-zone lymphoma according to the REAL
classification.5
Identification of a complex t(2;9;14)(p12;p13;q32) translocation in
the MZL-1 lymphoma.
Cytogenetic analysis of bone marrow cells of patient MZL-1 showed four
visible chromosomal abnormalities (for complete karyotype see Materials
and Methods). A partial duplication was observed on chromosome 1 [dupl(1)(q12q32)], a deletion on chromosome 7 [del(7)(q22q33)], a nonreciprocal translocation on
chromosome 17 [der(17)t(12;17)(q21;p12)], and a complex reciprocal
translocation involving chromosomes 2, 9, and 14 [t(2;9;14)(p12;p13;q32); Fig 3].
Interestingly, the Ig, PAX-5, and IgH genes
are located on 2p12,26 9p13,27 and
14q32,7 respectively, suggesting that these genes may have
participated in a three-way translocation during the genesis of the
MZL-1 lymphoma. Because the transcriptional polarity of the three genes
on the respective chromosomes is known,8,26 their
involvement in the complex t(2;9;14) translocation makes the following
prediction, which was subsequently verified by molecular analysis of
the breakpoint sequences (see below). The IgH gene has been
translocated in opposite orientation next to the Ig and
PAX-5 genes on the derivative chromosomes der(2) and der(14), respectively, whereas the Ig and PAX-5 loci have
been juxtaposed in the same transcriptional direction on chromosome
der(9).
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| Fig 3.
Cytogenetic anallysis of the complex t(2;9;14)
translocation of patient MZL-1. (A) Partial karyotype of three
representative metaphase nuclei. The normal homologues (left) and the
derivative (der) chromosomes (right) of the t(2;9;14)(p12;p13;q32)
translocation are shown in their G-banded form. (B) Schematic
representation of the t(2;9;14) translocation. Ideograms of the normal
and derivative chromosomes are shown together with the location and
transcriptional direction of the Ig, IgH, and
PAX-5 genes.
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Replacement of the endogenous PAX-5 promoters by a switch
Sµ promoter of the IgH locus in the MZL-1 translocation.
For Southern blot screening we chose a strategy that allowed us to
analyze lymphoma DNA for translocation breakpoints within 22 kb of the
PAX-5 5 region (from 6 to +16 kb relative to the transcription start of exon 1A; Fig 4). The
result of such an analysis is shown in Fig 4 for patients MZL-1 and
PLL-1. DNA isolated from MNC of these two patients was digested with
HindIII or EcoRI followed by Southern blot
hybridization with three different DNA probes. Whereas the exon
1A-specific probe #1 detected only the predicted DNA fragments of the
PAX-5 locus, a novel DNA fragment was observed with the exon
1B-specific probe #2 in each restriction digest of the MZL-1 DNA
compared with the `control' PLL-1 DNA. Interestingly, the adjacent
intron probe #3 detected extra HindIII and EcoRI
fragments of different lengths in the MZL-1 DNA. These data therefore
indicate that the translocation breakpoint in patient MZL-1 must reside
within the 3 end of PAX-5 exon 1B, most likely between
the DNA probes #2 and #3. Hence, each of the two DNA probes is specific
for one of the reciprocal PAX-5 translocation events. Moreover,
quantitation of the hybridization signals showed that the translocated
DNA fragment corresponded to ~80% of the wild-type PAX-5
DNA, thus confirming that the majority of the MNC of patient MZL-1
consisted of malignant B lymphocytes, in agreement with the flow
cytometric data (Fig 1A).
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| Fig 4.
Localization of the MZL-1 translocation breakpoint in
exon 1B of PAX-5. DNA from mononuclear cells of patients MZL-1
and PLL-1 was digested with HindIII or EcoRI
followed by Southern blot analysis with the indicated DNA probes. The
sizes of the hybridizing DNA fragments are indicated in kb to the left.
The DNA fragments containing the reciprocal translocation breakpoints
are highlighted in black. X denotes an unrelated DNA fragment that
cross-hybridizes with the GC-rich DNA probe #2. A restriction map of
the 5 region of the PAX-5 locus8 is shown
below together with the origin of the DNA probes and the hybridizing
HindIII and EcoRI DNA fragments (sizes given in
kb). Abbreviations: H, HindIII; E, EcoRI;
S, SacI; BS, BstEII; P,
PstI.
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To identify the partner genes involved in the MZL-1 translocation, we
took advantage of the fact that the two HindIII fragments containing the reciprocal PAX-5 translocation events were of
similar size (8.6 and 8.9 kb; Fig 4). We therefore used 8 to 9 kb
size-selected HindIII fragments of MZL-1 DNA to generate a
genomic phage library that was subsequently screened with the DNA
probes #2 and #3. Sequence analysis of the cloned HindIII
fragments showed that the reciprocal MZL-1 translocations are composed
of the gene arrangements shown in Fig 5. As
predicted by the cytogenetic analysis (Fig 3), the PAX-5 and
IgH genes are arranged in a head-to-head configuration on the
8.9-kb HindIII fragment isolated from the derivative chromosome 14 (Fig 5A). In agreement with the Southern blot data (Fig
4), the breakpoint was located in the 3 region of exon 1B of
the PAX-5 gene and in the 5 part of the switch Sµ
region of the IgH locus (Figs
5A and 6B). Interestingly, the Sµ region of the human IgH gene has been shown to harbor two antisense promoters,
which have been occasionally juxtaposed in a similar head-to-head
translocation next to the c-MYC gene in Burkitt's lymphoma,
thus resulting in the synthesis of a hybrid Sµ/c-MYC
transcript.28 The breakpoint of the MZL-1 translocation was
mapped ~70 nucleotides downstream of the heterogeneous start sites of
the second antisense Sµ promoter. Hence, in the MZL-1 translocation,
the PAX-5 gene on the derivative chromosome 14 was severed from
its own promoters and brought under the control of immunoglobulin Sµ
promoters.
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| Fig 5.
The breakpoints of the reciprocal PAX-5
translocations in patient MZL-1. The two translocation breakpoints on
the derivative chromosomes 14 (A) and 9 (B) were cloned as
HindIII DNA fragments and sequenced. Only the relevant
sequences across the breakpoints are shown below a schematic diagram of
each translocation event. Numbers refer to the corresponding nucleotide
positions of the PAX-5 exon 1B (Fig 6B) or immunoglobulin Sµ
sequence,28 respectively. Arrows denote the transcription
start sites of the antisense promoter in the immunoglobulin Sµ region
as determined by Apel et al.28
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| Fig 6.
Characterization of exon 1B of PAX-5. (A)
Heterogeneous transcription initiation. Total RNA (20 µg) isolated
from the B-lymphoid BJA-B and KIS-1 cell lines as well as from control
HeLa cells was analyzed by S1 nuclease protection assay with a DNA
probe labeled at the EagI site in exon 1B. The S1-resistant DNA
fragments were separated by electrophoresis together with a G+A
sequence ladder obtained by partial depurination of the same
end-labeled DNA probe. Arrowheads point to the most abundant
transcription start sites. (B) DNA sequence of exon 1B. The most
frequently used transcription start sites are denoted by arrows. Exon
1B sequences are shown in capital letters, whereas the flanking
promoter and intron sequences are shown in lowercase letters. The
numbers to the left refer to the nucleotide position relative to the
first prominent transcription start site. The deduced amino acids of
exon 1B, the reciprocal breakpoints [der(9) and der(14)] of the MLZ-1
translocation and the restriction sites relevant for probe design are
indicated together with a consensus recognition sequence for the
transcription factor Sp155 that was shown, by EMSA assay,
to bind to this 40 promoter region in B-cell nuclear extract (data
not shown). The invariant GT dinucleotide of the 5 splice
junction is underlined.
|
|
The gene configuration on the derivative chromosome 9 was also
consistent with the result of the cytogenetic analysis (Fig 3), because
the two PAX-5 promoters were positioned in the same transcriptional orientation next to the J segments of the
Ig gene (Fig 5B). This gene arrangement is, however,
unlikely to give rise to a functional transcription unit because the
translation start codon and the 3 splice site have been
eliminated from PAX-5 exon 1B during the translocation process.
Surprisingly, the PAX-5 and Ig genes were separated
by a 162 bp long sequence originating from the switch Sµ region of
the IgH locus (Fig 5B). This finding suggests that the complex
t(2;9;14)(p12;p13;q32) translocation has been brought about by two
consecutive rearrangements, each involving the Sµ sequences of the
IgH gene. The first reciprocal translocation must have occurred
between the PAX-5 and IgH genes, because the
alternative possibility, translocation between the IgH and
Ig loci, would have created a nonviable, dicentric
chromosome (Fig 3B). Subsequently, the translocated Sµ sequences on
the der(9) chromosome must have participated in a second rearrangement
involving the Ig gene. The involvement of the Sµ region in
both translocations strongly argues that the complex t(2;9;14)
translocation has been generated by misguided class-switch
recombination. In agreement with this idea, MZL has been postulated to
arise from activated B cells that normally undergo immunoglobulin class
switching.5 Moreover, errors of switch recombination have
previously been implicated as a major cause for translocations in
mature B-cell malignancies.29,30 Furthermore, it is
worthwhile to note that the reciprocal translocation between the
PAX-5 and IgH genes was accompanied by the loss of 163 and 215 bp from exon 1B and the Sµ region, respectively (Figs 5 and
6B). Hence, the MZL-1 translocation is not balanced at the fine
structural level in agreement with similar analyses of other
leukemias.31
Deregulation of PAX-5 expression by IgH control
elements in B lymphocytes carrying t(9;14) translocations.
In a next step, we investigated the transcription efficiency of the
wild-type and translocated PAX-5 alleles in B lymphocytes carrying t(9;14) translocations. The PAX-5 gene has been shown to be transcribed from two distinct promoters.8
Transcription from the upstream exon 1A promoter is initiated from a
single site 3 of a conserved
TATA-box.8 In contrast, transcription from
the downstream exon 1B promoter is heterogeneously initiated in the
B-lymphoid cell lines BJA-B and KIS-1, as shown by S1 nuclease protection analysis (Fig 6A). In agreement with this
finding, the downstream promoter of PAX-5 lacks any TATA-like
sequence, but instead contains a functional binding site for the
transcription factor Sp1 in the 40 region (Fig 6B). Sequence
analysis of exon 1B, furthermore, showed an extraordinarily long leader
region of 1.1-kb size (Fig 6B).
Two different types of B cells were available for studying the effect
of the t(9;14)(p13;q32) translocation on PAX-5 transcription, namely the MNC of patient MZL-1 and the KIS-1 cell line that was established from a diffuse large-cell lymphoma.19 In KIS-1
cells, the Eµ enhancer of the IgH locus was inserted upstream
of the exon 1A promoter of PAX-5,8 whereas the
immunoglobulin Sµ promoters replaced the downstream PAX-5
promoter in the B lymphocytes of patient MZL-1 (Fig 5A). To quantitate
the transcripts originating from the different promoters, we generated
riboprobes that were specific for the upstream PAX-5 promoter
(Ex1A), the downstream PAX-5 promoter (Ex1B), and the
immunoglobulin Sµ promoter (Sµ/Ex1B; Fig 7). In addition, we used a
riboprobe (Ex2-4) derived from the common coding sequences of exons 2-4 to map the total amount of PAX-5 mRNA. The results of the
various RNase protection analyses are shown in Fig 7. The PAX-5
transcript levels in the different B cells were quantitated and
normalized to the expression of the small ribosomal protein S16
gene.32 In parallel, the B-cell lines BJA-B and Raji as
well as primary B lymphocytes from two B-CLL patients were analyzed as
control cells, indicating that PAX-5 is expressed at a constant
level in these B lymphocytes in agreement with previous
analyses.12,13 Compared with these control cells, the
transcription from the upstream PAX-5 promoter was 8.3-fold
increased in the KIS-1 cells, which reflects the close proximity of the
Eµ enhancer to the exon 1A promoter in this lymphoma (Fig 7A). In
contrast, the downstream PAX-5 promoter was upregulated to a
lower degree (Fig 7B). Taking the transcription from both promoters and
mRNA splicing into account, we observed a 5.5-fold overexpression of
total PAX-5 mRNA in KIS-1 cells (Fig 7C). In comparison, the
PAX-5 mRNA level was 8.4-fold increased in the B lymphocytes of
patient MZL-1 (Fig 7C) as a result of efficient heterogeneous initation
of transcription at the Sµ promoter (Fig 7D). Hence, the two
different t(9;14) translocations analyzed did indeed lead to
deregulation of PAX-5 transcription by bringing the
PAX-5 gene under the control of immunoglobulin regulatory elements.
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| Fig 7.
Deregulation of PAX-5 transcription by t(9;14)
translocations. PAX-5 transcript levels were quantitated by
RNase protection assay in total RNA isolated from mononuclear blood
cells of patient MZL-1, from the large-cell lymphoma cell line KIS-1,
from the B-cell lines Raji and BJA-B, from mononuclear blood cells of
two patients diagnosed with B-CLL and from control HeLa cells. The
5 ends of the PAX-5A (A) and PAX-5B (B)
transcripts, the spliced mRNA sequences of the downstream paired domain
(C) and the hybrid Sµ-PAX5B transcripts (D) were mapped with
the riboprobes depicted below. The size of the expected RNase-protected
fragments is given in numbers of nucleotides. pUC19 DNA digested with
MspI was used as end-labeled DNA size marker (lane M; sizes
given in nucleotides). Transcripts coding for the small ribosomal
protein S1632 were comapped and used as an internal
reference for quantitation of the PAX-5 RNA signals by
PhosphorImager analysis. The induction of the different PAX-5
transcripts in the KIS-1 and MZL-1 cells was calculated relative to the
average PAX-5 expression level of the two B cell lines Raji and
BJA-B and is shown below the relevant part of the S16 autoradiograph.
The Sµ promoter lacks a TATA-box28 and thus results in
heterogeneous transcription initiation in MZL-1 cells (D).
Abbreviations: E, EcoRI; B, BamHI; EA,
EagI; S, Sau3A; P, PstI.
|
|
We next investigated whether the increased PAX-5 mRNA
production was also reflected at the protein level. For this purpose, the amount of BSAP (Pax-5) protein was quantitated by the sensitive EMSA method in nuclear extracts prepared from the different types of B
cells (Fig 8). A moderate, but consistent
2-2.5-fold increase in BSAP protein synthesis was observed in the KIS-1
and MZL-1 B lymphocytes as compared with the control BJA-B and Raji
cells. The discrepancy between the level of mRNA and DNA-binding
activity may indicate that PAX-5 expression is subject to
translational or posttranslational control in B cells.
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| Fig 8.
Moderate increase of BSAP protein synthesis in B
lymphocytes carrying the t(9;14) translocation. The same amount (1 µg) of nuclear protein isolated from mononuclear cells of patient
MLZ-1 or the indicated B-lymphoid cell lines was analyzed by EMSA assay
for binding of BSAP to the paired domain recognition sequence of the
H2A-2.2 gene. The radioactivity in the BSAP-DNA complex was
quantitated by PhosphorImager analysis, and the BSAP expression level
of the KIS-1 and MZL-1 cells is given relative to that of the control B
cell lines BJA-B and Raji. PAX-5 is not expressed in the
myeloma cell line RPMI 8226.
|
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Influence of the t(9;14) translocation on the expression of putative
BSAP target genes.
Targeted inactivation of the mouse Pax-5 gene has provided an
elegant genetic tool for dissecting the function of BSAP (Pax-5) in
gene regulation during early pro-B-cell development.14,15 In contrast, little is known about the role of BSAP during the late
phase of B-cell differentiation that has so far been inaccessible to
loss-of-function analysis. Protein-DNA binding and
transient-transfection experiments have, however, implicated BSAP in
the repression of the murine J-chain gene33 and in the
downmodulation of the IgH and Ig 3 enhancers
at the mature B-cell stage.10 The gain-of-function mutations of the human t(9;14)(p13;q32) translocations have now offered
an alternative approach for investigating the role of BSAP in gene
regulation at late developmental stages. The J-chain gene is known to
code for an immunoglobulin joining (J) protein that is essential for
the assembly and secretion of pentamer IgM antibodies.34 In
agreement with previous expression analyses,35 RNase
protection experiments showed that the human J-chain gene is broadly
expressed in a range of B-cell and myeloma cell lines (Fig 9A). Moreover, the J-chain gene was
also highly expressed in MNC of a healthy donor. In contrast,
transcripts of the J-chain gene could neither be detected in KIS-1
cells nor in MNC of the patient MZL-1 (Fig 9A). Hence, these data
indicate that overexpression of BSAP (Pax-5) is associated with
repression of J-chain gene transcription in B lymphocytes carrying the
t(9;14) translocation.
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| Fig 9.
J-chain and p53 gene expression in KIS-1 and
MZL-1 lymphomas. (A) Absence of J-chain gene expression in B
lymphocytes carrying the t(9;14) translocation. Human B-cell lines
(BJA-B, Raji, Ramos, HS-Sultan), myeloma cell lines (U226, RPMI 8226),
KIS-1 cells and mononuclear blood cells of patient MZL-1 as well as of
a control individual (MNC) were analyzed by RNase protection assay for
J-chain and S16 mRNA expression. (B) Normal p53
expression in tumors overexpressing PAX-5. The p53 mRNA
levels were quantitated by RNase protection analysis in a subset of the
cells studied in panel A. MB-238 refers to the tumor that exhibits the
highest PAX-5 expression level within a large medulloblastoma
collection.20 The KIS-1 cells carry a mutation resulting in
a truncated p53 mRNA (p53 ) that is detected on the longer
autoradiographic exposure shown to the left.
|
|
Recently, Stuart et al36 have proposed on the basis of
transient cell transfection data that Pax-5 (BSAP) is a potent
repressor of p53 gene transcription. According to this
hypothesis, Pax-5 should mediate its oncogenic effect through
inactivation of the tumor-suppressor gene p53. However, the
malignant B lymphocytes of patient MZL-1 expressed normal levels of
p53 mRNA compared with the control cell lines Raji, BJA-B, and
HeLa (Fig 9B). No intact p53 transcripts could be detected in
KIS-1 cells. However, a truncated p53 mRNA was expressed at a
low level, suggesting that both alleles of the p53 gene are
mutated in the KIS-1 cell line. Moreover, a medulloblastoma tumor,
MB-238, also contained normal levels of p53 mRNA (Fig 9B)
despite the fact that this tumor was previously shown to highly
overexpress the PAX-5 gene.20 Hence, these data do
not support a role for Pax-5 (BSAP) in p53 gene repression.
| |
DISCUSSION |
The transcription factor Pax-5 (BSAP) is known to play an important
role in early B-cell development as shown by loss-of-function mutation
in the mouse.11,14 The chromosomal t(9;14)(p13;q32) translocation has now been shown to correspond to a gain-of-function mutation of the human PAX-5 gene, pointing to a critical role of this transcription factor in late B-cell differentiation and in the
pathogenesis of NHL. As summarized in Fig
10, to date, three t(9;14) translocations have been molecularly
characterized, all of which affect the 5 region of the
PAX-5 gene. Recently, we have shown that the Eµ enhancer of
the IgH locus is juxtaposed next to the exon 1A promoter of
PAX-5 in the diffuse large-cell lymphoma KIS-1.8
Here, we have shown that an antisense promoter in the switch Sµ
region of the IgH gene is linked to the downstream exon 1B of
PAX-5 in a case of MZL. Interestingly, the translocation breakpoint of the B-SLL case 10529 was also located in
PAX-5 exon 1B just three base pairs upstream of that of patient
MZL-1 (Fig 5A and 10). The corresponding breakpoint on the IgH
locus was, however, only approximately mapped to the Sµ region, and
the effect of the t(9;14) translocation on PAX-5 expression was
not analyzed.9 Hence, it is unclear at present whether the
translocation in case 1052 occurred upstream or downstream of the
antisense Sµ promoter used in patient MZL-1. The same Sµ promoter
was previously shown to drive expression of a translocated c-MYC gene in Burkitt's lymphoma.28 In this tumor,
the coding region of the affected c-MYC gene is known to be
frequently altered by somatic mutations.37 Sequence
analysis of cloned PAX-5 cDNA from the MNC of patient MZL-1
demonstrated, however, that the translocated PAX-5 gene codes
for a wild-type BSAP protein (data not shown). Furthermore, expression
analyses directly showed an increase in the initiation of PAX-5
transcription from the Sµ promoter in the MZL-1 lymphoma and from the
upstream exon 1A promoter in the KIS-1 cells. These data indicate
therefore that the t(9;14) translocation corresponds to a regulatory
mutation that increases PAX-5 expression as a result of either
promoter replacement or enhancer insertion. In this regard, the
oncogenic activation of PAX-5 clearly differs from that of
PAX-3 and PAX-7 in alveolar rhabdomyosarcoma. A novel
fusion gene coding for a more potent transcription factor is generated
in this pediatric muscle tumor by a specific translocation involving
one of the two PAX loci and the fork head domain gene
FKHR.38-40
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| Fig 10.
t(9:14) translocations involving the PAX-5 and
IgH loci. A schematic diagram depicts the 5 region of
the PAX-5 gene, the JH-to-Cµ region
of the IgH locus and the corresponding translocation
breakpoints present on the derivative chromosme 14 in the lymphomas of
patients KIS-1,8 1052,9 and MZL-1 (this study).
The PAX-5 expression level and the translocation breakpoint
relative to the antisense promoters in the immunoglobulin Sµ region
have not been mapped in patient 1025,9 and hence it is not
known at present whether promoter replacement as described for patient
MZL-1 may be a molecular mechanism for PAX-5 gene deregulation
in this case. The position of the translation start codon at the very
3 end of PAX-5 exon 1B is indicated by ATG.
|
|
Southern blot analysis of 18 B-SLL tumors with plasmacytoid
differentiation did not show any further translocation in the promoter
regions of the PAX-5 gene. Hence, our analysis indicates, in
agreement with the study of Iida et al,9 that the immediate 5 region of PAX-5 is not a mutational hotspot in B-SLL
tumors. However, long-range FISH analysis has shown a more consistent involvement of PAX-5 in t(9;14) translocations of these
tumors,9 suggesting that activation from distantly located
IgH enhancers, as exemplified by the MAL
translocation,41 may be the most common mechanism for
deregulating the PAX-5 gene. As shown in the present report,
oncogenic activation of PAX-5 is not only observed in B-SLL
tumors with plasmacytoid differentiation, but also at least in one case
of MZL, a related non-Hodgkin's disease. The tumor of patient MZL-1
was classified as splenic MZL based on the observed splenomegaly, the
characteristic bone marrow infiltation pattern,16 the tumor
involvement of peripheral blood, the immunophenotype and villous
morphology of the malignant B lymphocytes,5 and the
expression of the DBA.44 antigen that is normally present only on cells
of the mantle and marginal zones of lymphoid follicles.25 Because the marginal zone is known to be enriched in memory B cells
with the potential of differentiating into plasma
cells,42,43 it has been postulated that transformation of
these cells may give rise to MZL.5
Recently, it has been suggested that Pax proteins exert their oncogenic
effect by inactivating the transcription of the tumor suppressor gene
p53.36 This hypothesis is primarily based on protein-DNA binding studies and transient cell transfection experiments that suggested that Pax-5 (BSAP) may repress the p53 gene by
binding to its 5 untranslated sequence.36 However,
we have been unable to confirm these data either in vitro or in vivo.
In contrast to the prediction of the hypothesis, the level of
p53 transcription was totally unaffected in early pro-B cells
lacking Pax-5.15 Second, we have not observed any
inverse correlation between PAX-5 and p53 expression in
a large panel of medulloblastoma tumors (Fig 9B; Z. Kozmik and M.B.,
unpublished data), which were previously shown to
overexpress the PAX-5 gene.20 Third, the
p53 expression level was also normal in the malignant B
lympocytes of patient MZL-1 (Fig 9B). Moreover, the previously reported
decrease of p53 expression in the large-cell lymphoma
KIS-19 was shown to be caused by a truncation of the
p53 transcript (Fig 9B) in agreement with the consistent
involvement of p53 gene mutations in disease progression to
large-cell lymphoma.44,45 All of these data therefore
strongly argue against the hypothesis that p53 is a target gene
of the transcription factor BSAP (Pax-5). In this context it is
interesting to note that the immunoglobulin J-chain gene was expressed
in all human B-cell lines analyzed except in the KIS-1 and MZL-1 B
lymphocytes. Hence, it is conceivable that overexpression of
PAX-5 may interfere with the regulation of the human J-chain
gene in analogy to the murine homologue that is considered to be under
negative control by BSAP.33
How can we explain the oncogenic activation of PAX-5 by the
t(9;14) translocation now that our data rule out an involvement of
PAX-5 in p53 gene regulation? In principle, we can
distinguish between a quantitative and temporal effect of PAX-5
deregulation on late B-cell differentiation. It has been well
documented that the function of PAX genes is highly sensitive
to gene dosage. This sensitivity is reflected by the frequent
association of heterozygous PAX gene mutations with human
disease syndromes46 as is best illustrated for
PAX-6. Inactivation of one PAX-6 allele causes aniridia
in humans and the Small eye phenotype in mice,47
whereas even a moderate increase in gene-copy number also causes a
Small eye-like phenotype referred to as
microphthalmia.48 By analogy, it is therefore possible that
the small, but consistent increase in BSAP (Pax-5) protein synthesis
could also alter the B-cell phenotype. For instance, antisense
oligonucleotide inhibition experiments suggested a role for BSAP in the
proliferation control of mature B cells.49 Interestingly,
the proliferation rate of these cells was even slightly increased by
transient overexpression of BSAP.49 Hence, it is
conceivable that deregulation of PAX-5 perturbs the cell-cycle
regulation of mature B cells, may prevent them from entering the
quiescent state, and could thus contribute to lymphomagenesis. On the
other hand, it is also possible that the proximity of immunoglobulin
control elements overrules the correct temporal regulation of
PAX-5 during the terminal phase of B-cell differentiation. The
PAX-5 gene is known to be downregulated during plasma cell
differentiation based on the following evidence. First, PAX-5
expression is detected in neither plasmacytoma nor myeloma cells of
mouse and human origin.12,50 Second, in vitro differentiation experiments with mouse splenic B lymphocytes have shown
that Pax-5 expression is repressed on stimulation of mature B
cells to undergo plasma cell differentiation.51,52 In
contrast, the IgH locus is transcriptionally most active in
immunoglobulin-secreting plasma cells.53 Hence, the
insertion of IgH control elements by the t(9;14) translocation
may force the translocated PAX-5 allele to remain active at a
time when the endogenous PAX-5 gene is usually switched off. In
agreement with this hypothesis, B lymphocytes carrying the t(9;14)
translocation frequently exhibit features of plasmacytoid
differentiation.4,9 Furthermore, ectopic expression of
PAX-5 in late B cells has recently been shown to interfere with
immunoglobulin secretion that is normally induced on terminal
differentiation.54 Hence, the t(9;14) translocation could
contribute to tumorigenesis by interfering with the downregulation of
PAX-5 transcription, thereby preventing completion of the
plasma cell differentiation program. This hypothesis can now be
experimentally verified by recreating the human t(9;14) translocation
breakpoint in transgenic mice.
| |
ACKNOWLEDGMENT |
We thank H. Ohno for providing the KIS-1 cell line, H. Pirc-Danoewinata
for cytogenetic analysis, R. Thalhammer for help with photography, R. Kurzbauer for DNA sequencing, P. Steinlein for cell sorting, and G. Christofori for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted March 3, 1998;
accepted July 10, 1998.
Supported by the Research Institute of Molecular Pathology, Vienna, by
a grant from the Austrian Industrial Research Promotion Fund, Vienna,
and by Grant No. NB5829 from the Austrian National Bank.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Meinrad Busslinger, PhD,
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030,
Vienna, Austria; email: Busslinger{at}nt.imp.univie.ac.at.
| |
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Abstract
Introduction
