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Blood, 15 April 2002, Vol. 99, No. 8, pp. 3060-3062
BRIEF REPORT
Loss of PU.1 expression is associated with defective
immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of
classical Hodgkin disease
Franziska Jundt,
Katharina Kley,
Ioannis Anagnostopoulos,
Kristina Schulze
Pröbsting,
Axel Greiner,
Stephan Mathas,
Claus Scheidereit,
Thomas Wirth,
Harald Stein, and
Bernd Dörken
From the Charité, Robert-Rössle-Klinik,
Humboldt University of Berlin, Germany; Max Delbrück Center for
Molecular Medicine, Berlin, Germany; Institute of Pathology, Klinikum
Benjamin Franklin, Free University of Berlin, Germany; Institute of
Pathology, University of Würzburg, Germany; Department of
Physiological Chemistry, University of Ulm, Germany
 |
Abstract |
Immunoglobulin transcription is impaired in Hodgkin and
Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD). We recently demonstrated that defective immunoglobulin promoter
transcription correlates with the down-regulation of the B-cell
transcription factors Oct2 and BOB.1/OBF.1. These results prompted us
to investigate whether immunoglobulin enhancer activity is also
impaired in HRS cells and whether as yet unidentified factors could be
necessary for immunoglobulin enhancer activity in HRS cells of cHD.
Here we analyzed 30 cases of cHD for expression of the Ets family
member PU.1 that is known to collaborate with multiple transcription factors and to regulate expression of immunoglobulin genes. We show
that PU.1 is not expressed in primary and cultured HRS cells. Reintroduction of PU.1 and Oct2 in cultured HRS cells restored the
activity of cotransduced immunoglobulin enhancer constructs. Our study
identifies PU.1 deficiency as a recurrent defect in HRS cells that
might contribute to their impairment of immunoglobulin transcription.
(Blood. 2002;99:3060-3062)
© 2002 by The American Society of Hematology.
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Introduction |
Absent immunoglobulin expression despite the
presence of clonal immunoglobulin gene rearrangements is a hallmark of
tumor cells of classical Hodgkin disease (cHD).1 Recently,
we showed that crippling mutations in the immunoglobulin coding region
and in the octamer motif of the immunoglobulin promoter are rare events in cHD.1 We further demonstrated that defective
immunoglobulin promoter transcription is most likely due to the
down-regulated expression of B-cell-specific transcription factors
such as Oct2 and BOB.1/OBF.1.1-3 In this study, we
investigated whether immunoglobulin enhancer activity is also impaired
in Hodgkin and Reed-Sternberg (HRS) cells and whether as yet
unidentified factors could be necessary for immunoglobulin enhancer
activity in HRS cells of cHD. We, therefore, analyzed the expression of
the Ets family transcription factor PU.1/SPI-1 that is known to
essentially contribute to the regulation of immunoglobulin-µ, - ,
and - gene expression.4
PU.1 is a tissue-restricted transcriptional regulator within the
hematopoietic system that is essential for the development of both B
cells and macrophages.4-9 Furthermore, several DNA-binding studies revealed that PU.1 binds to a large number of promoters and
regulates the expression of genes that are required for terminal differentiation of B cells.9,10 The list includes genes
encoding immunoglobulin light ( or ) and heavy chain genes,
RAG-1, immunoglobulin  (Ig-), Ig- , Vpre-B, 5, CD19, and
J-chain.5,10-14 Thereby, PU.1 requires interactions with
other transcription factors to regulate transcription and plays an
architectural role in forming higher-order complexes.15-17
Here we show that PU.1 is not detectable in cultured and primary HRS
cells of cHD. We demonstrate that cotransfection of PU.1 and Oct2 in
transient transfection studies activates immunoglobulin heavy chain
(IgH) intronic enhancer expression in cultured HRS cells. Together with
the absence of Oct2 and BOB.1/OBF.1, PU.1 deficiency is an important
recurrent defect of HRS cells probably contributing to defective
immunoglobulin gene expression in cHD.
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Study design |
Formalin-fixed and paraffin-embedded lymph node specimens from
30 patients with the diagnosis of cHD of nodular sclerosis or mixed
cellularity subtype were retrieved from the files of the Reference
Center for Lymph Node Pathology and Hematopathology at the Institute of
Pathology, Klinikum Benjamin Franklin, Free University of Berlin,
Berlin, Germany. Activated lymphoid tissue from palatinal tonsils
served as control. Tissue sections (4 µm) were deparaffinized and
subjected to a brief high-temperature pretreatment with the use of a
pressure cooker and 0.01 M citrate buffer pH 6.0. The sections were
incubated with the anti-PU.1 monoclonal antibody (clone G148-74
purchased from Becton Dickinson Biosciences, Heidelberg, Germany).
Bound antibody was visualized by using the EnVision Kit (DAKO,
Glostrup, Denmark).
Cell extracts were prepared and quantitated as
described.18 The primary antibodies were anti-PU.1
(Santa Cruz, CA) and anti-Spi-B (M.C. Simon, Howard Hughes
Medical Institute, Philadelphia, PA) antibodies.
Electroporation was performed as recently described.2,3
The luciferase reporter plasmids driven by conalbumin promoter with or
without the MluI-HpaI fragment of the human IgH gene
intronic enhancer (pEcona-Luc and pcona-Luc) were provided by Dr T. Watanabe (Kyusyu University, Fukuoka, Japan19). Murine PU.1
complementary DNA was provided by Dr D. Tenen (Harvard Medical School,
Boston, MA) and cloned in a pCDNA-3.1 expression plasmid (Invitrogen, Groningen, The Netherlands).
| |
Results and discussion |
In this study we investigated the role of the B-cell transcription
factor and Ets family member PU.1 in defective immunoglobulin gene
expression in HRS cells of cHD. For this purpose we analyzed 30 cases
of cHD for expression of PU.1 by immunohistochemistry. In all
cases PU.1 expression was totally absent from HRS cells, whereas
we found, as expected, PU.1 protein levels in reactive B cells,
granulocytes, and macrophages surrounding HRS cells (Figure 1B). PU.1 expression was further detected
in the various B-cell compartments of reactive lymphoid tissues that
served as a positive control (Figure 1A). In contrast to our analysis
of the transcription factor Oct2 and its coactivator BOB.1/OBF.1, that
were partially present at low levels in 9.4% and 25% of cases of cHD
respectively,2 PU.1 expression was not detectable in any
case. PU.1 deficiency, therefore, is a recurrent defect of a B-cell
transcription factor in HRS cells of cHD, underlining our hypothesis
that absent immunoglobulin expression in cHD is probably due to defects
in the transcriptional machinery. These data are in accordance with our
recent results, showing that neither crippling mutations within
the V region gene nor in the octamer motif of the immunoglobulin
promoter or other regulatory sequences can be the general cause for the
defective immunoglobulin transcription in cHD.1-3
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| Figure 1.
Expression pattern of PU.1 in reactive lymphoid tissue and in classical
Hodgkin disease.
(A) Uniform nuclear expression of PU.1 by germinal center
and follicular mantle B cells in tonsillar lymphoid
tissue. (B) Hodgkin and Reed-Sternberg cells show absent PU.1
expression, whereas small B cells and occasional histiocytes express
the protein in the nuclei (immunostains using a PU.1 monoclonal
antibody and the EnVision method using diaminobenzidine as chromogen,
brown reaction product). Isotype-matched irrelevant antibodies did not
show positive staining patterns on the same tissues. Original
magnification A, × 100; B, × 250.
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To verify PU.1 deficiency in cultured HRS cells, we performed Western
blot analysis by using PU.1-specific antibodies (Figure 2A). As expected, all Hodgkin cell lines
(lanes 1-6) did not show PU.1 protein levels in contrast to other
B-cell-derived non-Hodgkin cell lines (lanes 7-9) and
CD19+ B cells (lane 10). Correlating with the absence of
PU.1 protein levels, we did not detect any PU.1-specific transcripts in
the Hodgkin cell lines by Northern blot analysis (data not shown). Whereas PU.1 messenger RNA was readily detectable in the non-Hodgkin cell lines tested (data not shown).
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| Figure 2.
PU.1 expression in Hodgkin cell lines and transient
cotransfections of the Hodgkin cell lines L428 and KM-H2 with
immunoglobulin enhancer constructs.
(A) Western blot analysis of cell lysate proteins from Hodgkin (lanes
1-6), non-Hodgkin (Burkitt lymphoma: Daudi, Namalwa; pre-B cell line:
Reh; lanes 7-9) cell lines and mature CD19+ B cells (B
cells; lane 10), showing bands of PU.1 and Spi-B. Left margin, size
markers in kilodalton. L428 (B) and KM-H2 (C) cells were transfected
with luciferase reporter plasmids driven by conalbumin promoter with or
without the MluI-HpaI fragment of the human IgH gene
intronic enhancer (pEcona and pcona). Expression vectors for
PU.1, Oct2, and BOB/OBF.1 were cotransfected as indicated. Relative
luciferase activity is shown and transfections with empty expression
vectors were arbitrarily set to 1.
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However, Spi-B, another member of the Ets family of DNA-binding
proteins,20,21 was expressed in several Hodgkin and
non-Hodgkin cell lines (Figure 2A; lanes 1, 2, 5-8) and in
CD19+ B cells (lane 10). Our data indicate that in contrast
to the Ets family member Spi-B, PU.1 is not expressed in cultured HRS cells. All tested Hodgkin cell lines, therefore, resemble tumor cells
in vivo with respect to PU.1 expression. The correspondence between our
in vitro and in vivo data is particularly important, because recent
studies of our group showed that there might be discrepant results
studying B-cell transcription factors in cultured and primary HRS
cells. High expression of Oct2, shown in cultured HRS cells by
electromobility shift assays, could subsequently neither be detected in
primary nor in cultured tumor cells by our very sensitive in situ
hybridization analysis.2,22 This discrepancy could
possibly be explained by cell culture conditions that might have
transitorily induced Oct2 levels in the Hodgkin cell lines.
There is abundant evidence that PU.1 is important for heavy and light
chain immunoglobulin gene transcription.10-14 PU.1
stimulates, for example, the activity of the immunoglobulin-
enhancer by playing an architectural role in the assembly of a
higher-order protein-DNA complex.17 Thereby binding of
phosphorylated PU.1 to its target sequence in the 3' enhancer results
in the cooperative recruitment of additional transcription factors such
as Pip, E2A, and AP-1 to adjacent DNA sites.17 However,
none of the enhancer binding proteins alone can significantly activate
the 3' enhancer.17 To investigate whether PU.1 can
contribute to immunoglobulin enhancer activity in cultured HRS cells,
we performed cotransfection experiments in which we reintroduced PU.1
and Oct2 as well as the Oct2 coactivator BOB.1/OBF.1 into L428 and
KM-H2 cells (Figure 2B,C). By using luciferase reporter constructs
driven by conalbumin promoter with the human IgH intronic enhancer, we
showed that in L428 and KM-H2 cells PU.1, Oct2, and BOB.1/OBF.1 alone
only slightly activated reporter constructs. However, reintroduction of
PU.1 together with Oct2 (L428 cells, Figure 2B; KM-H2 cells, Figure 2C)
significantly enhanced reporter activity. This activity was identical
to that obtained in the Burkitt lymphoma cell lines Daudi and Namalwa (data not shown), expressing endogenous levels of these factors (Figure
2A and data not shown). Our data indicate that in HRS cells PU.1 can
cooperate with Oct2 in activation of the human IgH gene intronic
enhancer. These data are in line with a previous report, suggesting
that PU.1 plays a general role for the activation of immunoglobulin
gene transcription.10 According to these data PU.1 can
bind to pyrimidine-rich motifs that are functionally important, in
particular for those regulatory elements that have only imperfect
octamer sites. PU.1 and Oct2 bind concomitantly to these elements and
thereby activate immunoglobulin gene transcription.10 Because there are binding sites in the IgH intronic enhancer for PU.1
and Oct2, it seems conceivable that they regulate enhancer activity in
our reporter constructs.
In our recent study we used reporter constructs with the wild-type or
mutated immunoglobulin promoter octamer motif.2 We demonstrated a high transcriptional activation of the immunoglobulin promoter construct after cotransfection of Oct2 and/or its coactivator BOB.1/OBF.1 in L428, KM-H2, and L1236 cells.2,3 We
conclude from our recent and present results that in HRS cells
dependent on the immunoglobulin promoter or enhancer context, Oct2,
BOB.1/OBF.1, and PU.1 can contribute to their transcriptional activation.
In summary, our data suggest that the loss of expression of several
important B-cell transcription factors such as Oct2, BOB.1/OBF.1, and
PU.1 is responsible for the inactivity of immunoglobulin promoters and
enhancers in HRS cells of cHD.
| |
Footnotes |
Submitted September 4, 2001; accepted November 30, 2001.
Supported by the Deutsche Forschungsgemeinschaft through JU 426/1-1.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Franziska Jundt, Charité,
Robert-Rössle-Klinik, Humboldt University of Berlin, D-13125
Berlin, Germany; e-mail: fjundt{at}mdc-berlin.de.
| |
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Top
Abstract
Introduction