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NEOPLASIA
From the Department of Pathology, VU University
Hospital, Amsterdam; and Swammerdam Institute for Life Sciences,
BioCentrum Amsterdam, University of Amsterdam, The Netherlands.
Polycomb-group (PcG) proteins, such as BMI-1 and EZH2, form
multimeric gene-repressing complexes involved in axial patterning, hematopoiesis, and cell cycle regulation. In addition, BMI-1 is involved in experimental lymphomagenesis. Little is known about its
role in human lymphomagenesis. Here, BMI-1 and EZH2 expression patterns are analyzed in a variety of B-cell non-Hodgkin lymphomas (B-NHLs), including small lymphocytic lymphoma, follicular lymphoma, large B-cell lymphoma, mantle-cell lymphoma, and Burkitt lymphoma. In
contrast to the mutually exclusive pattern of BMI-1 and EZH2 in
reactive follicles, the neoplastic cells in B-NHLs of intermediate- and
high-grade malignancy showed strong coexpression of BMI-1 and EZH2.
This pattern overlapped with the expression of Mib-1/Ki-67, a marker
for proliferation. Neoplastic cells in B-NHL of low-grade malignancy
were either BMI-1low/EZH2+ (neoplastic
centroblasts) or BMI-1lowEZH2 B-cell non-Hodgkin lymphomas (B-NHLs) are clonal
disorders with a mature phenotype and rearranged immunoglobulin
genes.1 These tumors show a wide spectrum of morphologic
features that vary between a nearly intact preservation of nodal
architecture in follicular lymphoma and a diffuse growth pattern in
most large B-cell lymphomas and Burkitt lymphoma.2-4 The
pathogenic mechanism leading to B-NHL is probably a multistep process
related to the inherent genetic instability associated with
immunoglobulin rearrangement,5 external factors such as
impaired or suppressed immunity, and a variety of environmental
factors.6
Polycomb-group (PcG) proteins play a role in body plan formation (axial
patterning through the repression of Hox genes),
hematopoiesis, and checkpoints affecting cell cycle
entry.7-11 Recent experiments also identified PcG proteins
as a group of gene-regulatory factors that may contribute to
oncogenesis and lymphomagenesis. PcG proteins form large, multimeric
complexes that bind to chromatin and probably function by altering
chromatin structure.12,13 Some PcG proteins may repress
gene activity through histone deacetylation.14 So far, 2 PcG complexes have been identified: a complex containing the ENX/EZH2
and EED PcG proteins and another complex consisting of BMI-1, RING1,
HPH1, HPH2, HPC1, HPC2, and HPC3.9,15,16 These complexes
are hypothesized to have opposing roles: predominance of one complex
may maintain cells in a proliferative state, whereas predominance of
the other complex is seen in differentiated cells.17-19 Previously, we demonstrated that the expression of PcG complexes during
germinal center (GC) reaction is linked to the differentiation status
of follicular B cells. We observed a mutually exclusive pattern of
BMI-1/RING1 and EZH2/EED PcG proteins in reactive centroblasts and
centrocytes. EZH2/EED expression was seen in dividing
centroblasts of GC dark zones, and BMI-1/RING1 expression was
dominant in resting B cells of the mantle zones and centrocytes in the
light zones.20,21 These observations suggested that the
expression of PcG complexes is highly regulated during GC reaction and
that PcG proteins may contribute to antigen-specific B-cell maturation.
Deregulation of PcG gene expression in experimental model
systems has clearly been linked to oncogenesis. For instance, the overexpression of Bmi-1 resulted in lymphomas in transgenic
mice.10 In addition, the overexpression of RING1 caused
anchorage-independent growth, cellular transformation, and metastatic
activity in nude mice.12 Yet, little is known about a
possible role for PcG genes in human lymphoma. We recently
demonstrated that Mib-1/Ki-67+ Hodgkin-Reed-Sternberg
(HRS) cells coexpress EZH2 and BMI-1.21 Because most HRS
cells originate from B cells in reactive follicles, where the
expression of BMI-1 and the expression of EZH2 are mutually exclusive, this pattern suggested that Hodgkin lymphoma is associated with deregulated expression of PcG complexes.
In the current study, we questioned whether B-NHL is also associated
with BMI-1/EZH2 coexpression in Mib-1/Ki-67-expressing neoplastic B
cells. Using unique antisera against BMI-1 and EZH2, we found
BMI-1/EZH2 coexpression in Mib-1/Ki-67+ neoplastic large
cells in intermediate- and high-grade B-NHL. Large
Mib-1/Ki-67+ neoplastic cells in low-grade B-NHL showed
weak coexpression of EZH2 and BMI-1. By contrast, small neoplastic
cells in low-grade B-NHL showed reduced BMI-1 expression in the absence
of EZH2 or Mib-1/Ki-67. We concluded that human B-NHL, such as Hodgkin
lymphoma, is associated with irregular expression of BMI-1 and EZH2
PcG genes. In addition, the level of BMI-1/EZH2 coexpression
correlated with clinical grade and the presence of Mib-1/Ki-67
expression. These findings suggest that the irregular expression of
BMI-1 and EZH2 is an early event in the formation of B-NHL, and they point to a role for abnormal PcG expression in human lymphomagenesis.
Patient material
Antibodies used in this study
Immunohistochemical detection of human PcG gene expression Expression of the BMI-1 and EZH2 PcG proteins was detected using the 6C9 mouse monoclonal antibody (anti-BMI-1) and the polyclonal K358 rabbit antiserum (anti-EZH2), respectively.13 After deparaffinization, endogenous peroxidase was inhibited by incubation of the tissue sections for 30 minutes at room temperature in 0.3% H2O2, diluted in methanol. Antigens were retrieved by boiling for 10 minutes in citrate buffer (pH, 6), followed by successive rinses in phosphate-buffered saline (PBS) containing 0.5% Triton (1 × 5 minutes) and then in PBS only (3 × 5 minutes). Slides were incubated for 10 minutes in 0.1 M glycine (diluted in PBS) and rinsed in PBS only (3 × 5 minutes). Before application of the primary antiserum or antibody, sections were incubated for 10 minutes in normal swine serum (diluted 1:10 in PBS + 1% BSA) or normal rabbit serum (diluted 1:50 in PBS + 1% BSA). Secondary antisera were biotinylated goat-antimouse or biotinylated swine-antirabbit (Dako, Glostrup, Denmark). Immunostaining was performed with 3-amino-9-ethylcarbazole using the streptavidin-biotin complex-horseradish peroxidase method (Dako) and tyramine intensification. Sections were counterstained with hematoxylin. Photographs were taken with a Zeiss Axiophot microscope and digitized using an Agfa duoscan scanner.Double immunofluorescence Tissue sections were fixed in 2% formaldehyde, and endogenous peroxidase was inhibited as above. After preincubation with 5% BSA, a combination of 2 primary antibodies was applied overnight at 4°C either anti-BMI-1 (6C9; mouse IgG2b monoclonal antibody) and
anti-EZH2 (K358; rabbit polyclonal antiserum) or EZH2 with anti-Ki-67
(MIB1; mouse IgG1 monoclonal antibody; Immunotech). BMI-1 or Ki-67 was
detected by biotinylated goat antimouse antiserum followed by
streptavidin-Cy3 (Immunoresearch, Jackson, PA), whereas EZH2 was
detected by swine-antirabbit Ig-fluorescein isothiocyanate (FITC;
Dako). Alternatively, green fluorescence was performed using
Alexa-linked goat-antirabbit immunoglobulin (Molecular Probes, Eugene,
OR). For each double-immunofluorescence experiment, single-color controls were included.
Coexpression of BMI-1, EZH2 in Mib-1/Ki-67+ neoplastic cells of large B-cell lymphoma We started our study of PcG expression in human B-NHL with an analysis of large B-cell lymphoma (including follicular lymphoma grade III either with or without a residual follicular growth pattern) and diffuse large B-cell lymphoma. Neoplastic centroblasts in these lymphomas showed clear nuclear staining for BMI-1 (Figure 1A), to an extent almost similar to that of EZH2 (Figure 1B). Staining in these large B-cell lymphomas appeared comparable to the pattern obtained for Mib-1/Ki-67 (Figure 1C). Using double immunofluorescence, we confirmed that neoplastic centroblasts expressed BMI-1 (red signal in Figure 1D) and EZH2 (green staining in Figure 1E) in the same nucleus (producing a yellow signal in Figure 1F). Note that normal cells in the surrounding infiltrate are BMI-1+/EZH2 (see Figure 10). In addition,
neoplastic cells expressed Mib-1/Ki-67 (red signal; Figure 1G) in
combination with EZH2 (green signal; Figure 1H), resulting in yellow
nuclear staining after combining the 2 signals (Figure 1I). From these
patterns, we concluded that coexpression of BMI-1 and EZH2 coincided
with cycling Mib-1/Ki-67+ neoplastic cells. We observed the
same pattern in nodal and extranodal large B-cell lymphoma (not shown).
Neoplastic Mib-1/Ki-67+ cells in mantle-cell and Burkitt lymphoma coexpress BMI-1 and EZH2 Mantle-cell lymphoma and Burkitt lymphoma showed an expression pattern of BMI-1, EZH2, and Mib-1/Ki-67 that closely resembled the expression profile of large B-cell lymphoma. In mantle-cell lymphoma, neoplastic cells showed both BMI-1 (correlating with reactive mantle cells) and EZH2 expression (shown as double immunofluorescence in Figure 2A). Almost all EZH2-expressing cells expressed Mib-1/Ki-67 (shown as double immunofluorescence in Figure 2B). Therefore, expression patterns of EZH2 and BMI-1 showed considerable overlap in mantle lymphoma cells (single colors not shown), comparable to the pattern in large B-cell lymphoma. Note that large green EZH2+ and Mib-1/Ki-67 cells are
also present: these are probably pre-existing blasts.
The pattern of BMI-1 and EZH2 in virtually all Burkitt lymphoma cells showed overlap similar to that of large B-cell blasts in B-NHL. Burkitt blasts coexpressed BMI-1 (red signal) and EZH2 (green signal), producing a yellow signal in Figure 2C. Overlap between EZH2+ neoplastic cells and Mib-1/Ki-67+ cells was observed for almost all Burkitt blasts (Figure 2D). Decreased BMI-1 expression in neoplastic centrocytes of low-grade follicular lymphoma Because the coexpression of BMI-1 and EZH2 correlated with cycling Mib-1/Ki-67+ cells in intermediate- and high-grade lymphomas, we subsequently analyzed the expression profile of these PcG proteins in low-grade B-NHL. Figure 3 shows representative immunohistochemical staining patterns for BMI-1 and EZH2 in follicular lymphoma. In control tissue, expression of BMI-1 (Figure 3A) and EZH2 (Figure 3B) was as determined previously.20,21 In general, mantle cells, intrafollicular macrophages, and light zone centrocytes in reactive follicles were positive for BMI-1. These cells did not stain for EZH2, which was mainly detectable in dark-zone centroblasts. In low-grade follicular lymphoma with preserved follicular architecture, we observed reduced BMI-1 staining in neoplastic centrocytes (Figure 3C, see legend) compared to the more intense staining pattern in surrounding infiltrating cells and centrocytes of reactive follicles (Figure 3A). EZH2 expression in low-grade follicle center lymphoma was confined to reactive and neoplastic centroblasts (Figure 3D; overview). In diffuse areas, small numbers of neoplastic centroblasts were observed (Figure 3E; detail) comparable to the number of Mib-1/Ki-67-expressing cells (Figure 3F). The pattern in small lymphocytic lymphoma was similar to that in low-grade follicular lymphoma (not shown). We concluded that BMI-1 expression is decreased in neoplastic centrocytes in follicular lymphoma and neoplastic small cells in small lymphocytic lymphoma. EZH2 expression in neoplastic centroblasts appeared unchanged compared to that in reactive centroblasts.
Weak coexpression of BMI-1 and EZH2 in neoplastic centroblasts of low-grade follicular lymphoma To determine whether EZH2+ neoplastic centroblasts in low-grade follicular lymphoma coexpress BMI-1, we analyzed the expression of these proteins with double immunofluorescence. BMI-1 was detected in neoplastic centrocytes of low-grade follicular lymphoma and neoplastic small lymphocytes in small lymphocytic lymphoma. As can be seen in Figure 4A, BMI-1 expression was seen throughout, both in neoplastic centrocytes and in neoplastic centroblasts. EZH2 expression was detected in a limited number of centroblasts (Figure 4B), and double immunofluorescence showed that large cells/blasts weakly expressed BMI-1 in the presence of strong EZH2 expression. This produced a yellowish hue in these centroblasts (Figure 4C). A similar pattern was observed in lymphoplasmacytoid lymphomaweak but detectable BMI-1 expression (Figure 4D) in small
numbers of EZH2-expressing neoplastic cells (Figure 4E), resulting in
weak BMI-1/EZH2 coexpression (Figure 4F). Note that coexpression of BMI-1 and EZH2 rarely occurs in normal reactive follicles, whereas BMI-1 and EZH2 expression in centroblasts and centrocytes is mutually exclusive (not shown, but discussed in detail
elsewhere).20,21
In follicular lymphoma Mib-1/Ki-67 and EZH2 expression overlapped (Figure 4G-I), similar in pattern to that in small lymphocytic lymphoma (not shown). We concluded from this pattern that Mib-1/Ki-67+ cells in low-grade B-NHL are cells that weakly coexpress BMI-1 and EZH2. In summary, all B-NHL tested showed aberrant PcG expression compared to reactive follicular cells. In general, small centrocytes and lymphocytes in low-grade follicular lymphoma and small lymphocytic lymphoma expressed BMI-1 at reduced levels compared to their reactive counterparts. In these lymphomas, EZH2 expression was limited to large Mib-1/Ki-67+ cells that weakly coexpress BMI-1. In contrast, neoplastic mantle cells, Burkitt cells, and blast cells in large-cell B-NHL showed strong double expression of BMI-1/EZH2 that always overlapped with Mib-1/Ki-67.
PcG genes encode a new class of gene regulatory factors that contribute to normal lymphoid development and lymphomagenesis. They were originally discovered in Drosophila, where they regulate embryonic development as inhibitors of homeobox gene expression. Polycomb proteins function by forming multimeric protein complexes that bind chromatin. Two fundamental complexes have been identified, but their composition can differ in various cell types. This variation is most likely related to target gene specificity9 and the role of PcG complexes in the maintenance of cellular identity during cell division. The 2 human PcG complexes are expressed at various stages of GC B-cell development.20,21 However, their expression depends on differentiation stage and stage in the cell cycle: dividing centroblasts express the complex identified by the EZH2 PcG protein, whereas resting mantle cells and centrocytes use the complex identified by BMI-1. BMI-1 and EZH2 are rarely detected in the same nucleus of follicular B cells, suggesting that expression of the 2 complexes is mutually exclusive and highly regulated. There is increasing evidence that the deregulation of PcG expression is related to the formation of lymphomas. A well-studied example is the Bmi-1 transgenic mouse, which exhibits increased lymphoproliferation and induction of lymphomas.10,25,26 We recently demonstrated that one malignant counterpart of follicular B cells, the HRS cell in Hodgkin lymphoma, coexpresses BMI-1 and EZH2.21 This suggested that deregulated PcG expression may be related to human lymphomagenesis as well. In the current study, we analyzed BMI-1 and EZH2 expression in various classes of B-NHL and questioned whether neoplastic B cells coexpress BMI-1 and EZH2. We observed 2 aberrant expression patterns of these PcG proteins. Tumor cells in intermediate- and high-grade B-NHL (large B-cell NHL, Burkitt lymphoma, and mantle-cell lymphoma) expressed BMI-1 at high levels (BMIhigh), virtually always in the presence of EZH2. By contrast, tumor cells in low-grade B-NHL (follicular lymphoma, small lymphocytic lymphoma) expressed low levels of BMI-1 (BMI-1low), either in the presence (neoplastic centroblasts) or absence (neoplastic centrocytes) of EZH2. Furthermore, the detection of EZH2 in B-NHL neoplastic cells overlapped with expression of the Mib-1/Ki-67+ proliferation marker. The irregular expression profile of BMI-1 and EZH2 in B-NHL suggests that the distinct balance between the BMI-1 and EZH2-containing PcG complex is disturbed in these lymphomas. Furthermore, the extent of irregular PcG expression correlated with the type of lymphoma (and, therefore, clinical behavior). Small neoplastic centrocytes in low-grade B-NHL exhibited decreased BMI-1 expression in the absence of EZH2, and larger neoplastic blasts in these low-grade B-NHL showed weak coexpression of BMI-1 and EZH2. By contrast, tumor cells in intermediate- and high-grade B-NHL were strongly positive for BMI-1 and EZH2. These results suggest that the balance between BMI-1 and EZH2 expression is progressively disturbed in dividing cells of intermediate- and high-grade B-NHL lymphomas. Because PcG complexes are involved in the maintenance of the cellular differentiation program, altered PcG expression patterns could at least partially explain the different behavior of neoplastic cells. Although our study does not resolve the mechanism that accounts for
BMI-1/EZH2 coexpression in neoplastic cells, the expression profile in
the normal counterparts of these cells allows us to speculate about a
possible mechanism. Expression of EZH2 in Mib-1/Ki-67+
neoplastic B cells appears to be natural, because normal
Mib-1/Ki-67+ follicular B cells express EZH2 as
well.20,21 In addition, in vitro up-regulation of EZH2
transcription has been reported during the entry of lymphocytes into
the cell cycle.27 We conclude that the aberrant PcG
expression pattern in B-NHL is related to the presence of BMI-1 in
dividing neoplastic cells, suggesting overexpression of this
PcG gene in neoplastic cells. Normal follicular B cells do
not express BMI-1 when they are dividing, and BMI-1 is only detected in
EZH2 One important aspect of PcG complex expression that should be addressed
in future studies is the fine composition of the complexes expressed in
normal and transformed cells. It is unknown whether PcG expression
patterns, as determined in Hodgkin lymphoma and B-NHL, represent
normally assembled PcG complexes. We recently found that
BMI-1+/RING1+/EZH2 In conclusion, we demonstrated that low-, intermediate-, and high-grade B-NHL are associated with increased coexpression of the BMI-1 and EZH2 PcG proteins, whose normal expression pattern is mutually exclusive. The underlying mechanism of this expression pattern is most likely related to a failure to down-regulate BMI-1 in dividing neoplastic cells, which is in agreement with observations in Bmi-1 transgenic mice. The extent of BMI-1/EZH2 coexpression correlated with clinical grade and the presence of Mib-1/Ki-67 expression. This suggests that the irregular expression of BMI-1 and EZH2 is an early event in the formation of B-NHL and points to a role for abnormal PcG expression in human lymphomagenesis.
Submitted August 21, 2000; accepted February 16, 2001.
F.J.v.K. and F.M.R. contributed equally to this study.
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: Folkert J. van Kemenade, Department of Pathology, VU University Hospital, Rm PA-001, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands; e-mail: f.vkemenade{at}azvu.nl.
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 V. K. Rajasekhar and M. Begemann Concise Review: Roles of Polycomb Group Proteins in Development and Disease: A Stem Cell Perspective |
Top
Abstract
Introduction
), as indicated in panels D and F. Note that small (infiltrating)
lymphocytes in panels D to F show expression of BMI-1 but not of EZH2,
as expected. (G-I, third row) IF of large B-cell lymphoma shows
expression of Mib/Ki67 in red (G) and EZH2 in green (H). Double
fluorescence confirms the coexpression of these proteins in the nuclei
of tumor cells (J, overlay photographic exposure with yellow nuclei).
Same example as in panels D to F is shown in panels G to I. All IF
pictures were taken with 63× objective.
