Differential expression of human Polycomb group proteins in various tissues and cell types.

Polycomb group proteins are involved in the maintenance of cellular identity. As multimeric complexes they repress cell type-specific sets of target genes. One model predicts that the composition of Polycomb group complexes determines the specificity for their target genes. To study this hypothesis, we analyzed the expression of Polycomb group genes in various human tissues using Northern blotting and immunohistochemistry. We found that Polycomb group expression varies greatly among tissues and even among specific cell types within a particular tissue. Variations in mRNA expression ranged from expression of all analyzed Polycomb group genes in the heart and testis to no detectable Polycomb group expression at all in bone marrow. Furthermore, each Polycomb group gene was expressed in a different number of tissues. RING1 was expressed in practically all tissues, while HPH1 was expressed in only a few tissues. Also within one tissue the level of Polycomb group expression varied greatly. Cell type-specific Polycomb group expression patterns were observed in thyroid, pancreas, and kidney. Finally, in various developmental stages of fetal kidney, different Polycomb group expression patterns were observed. We conclude that Polycomb group expression can vary depending on the tissue, cell type, and development stage. Polycomb group complexes can only be composed of the Polycomb group proteins that are expressed. This implies that with cell type-specific Polycomb group expression patterns, cell type-specific Polycomb group complexes exist. The fact that there are cell type-specific Polycomb group targets and cell type-specific Polycomb group complexes fits well with the hypothesis that the composition of Polycomb group complexes may determine their target specificity. J. Cell. Biochem. Suppl. 36: 129-143, 2001.

Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma.

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-1(low)/EZH2(+) (neoplastic centroblasts) or BMI-1(low)EZH2(-) (neoplastic centrocytes). These observations show that low-, intermediate-, and high grade B-NHLs are associated with increased coexpression of the BMI-1 and EZH2 PcG proteins, whose normal expression pattern is mutually exclusive. This expression pattern is probably caused by a failure to down-regulate BMI-1 in dividing neoplastic cells, because BMI-1 expression is absent from normal dividing B cells. These observations are in agreement with findings in studies of Bmi-1 transgenic mice. The extent of BMI-1/EZH2 coexpression correlated with clinical grade and the presence of Mib-1/Ki-67 expression, suggesting that the irregular expression of BMI-1 and EZH2 is an early event in the formation of B-NHL. This points to a role for abnormal PcG expression in human lymphomagenesis. (Blood. 2001;97:3896-3901)

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation.

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.

Coexpression of BMI-1 and EZH2 polycomb group genes in Reed-Sternberg cells of Hodgkin's disease.

The human BMI-1 and EZH2 polycomb group (PcG) proteins are constituents of two distinct complexes of PcG proteins with gene regulatory activity. PcG proteins ensure correct embryonic development by suppressing homeobox genes, and they also contribute to regulation of lymphopoiesis. The two PcG complexes are thought to regulate different target genes and probably have different tissue distributions. Altered expression of PcG genes is linked to transformation in cell lines and induction of tumors in mutant mice, but the role of PcG genes in human cancers is relatively unexplored. Using antisera specific for human PcG proteins, we used immunohistochemistry and immunofluorescence to detect BMI-1 and EZH2 PcG proteins in Reed-Sternberg cells of Hodgkin's disease (HRS). The expression patterns were compared to those in follicular lymphocytes of the lymph node, the normal counterparts of HRS cells. In the germinal center, expression of BMI-1 is restricted to resting Mib-1/Ki-67(-) centrocytes, whereas EZH2 expression is associated with dividing Mib-1/Ki-67(+) centroblasts. By contrast, HRS cells coexpress BMI-1, EZH2, and Mib-1/Ki-67. Because HRS cells are thought to originate from germinal center lymphocytes, these observations suggests that Hodgkin's disease is associated with coexpression of BMI-1 and EZH2 in HRS cells.

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers.

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.

High prevalence of codon 213Arg-->Stop mutations of the TP53 gene in human ovarian cancer in the southwestern part of The Netherlands.

As in many human malignancies, TP53 mutations are the most common genetic alterations in malignant human ovarian tumours. An approach often used in the determination of TP53 status is immunohistochemical staining of the protein. Non-missense mutations, especially those of the null type, causing premature termination codons and resulting in truncated proteins, may often not be detectable by immunohistochemistry. Therefore, current estimates of TP53 alterations in ovarian cancer may be inaccurate. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing techniques, we have found a high prevalence of TP53 non-missense mutations in exons 5-8 in ovarian tumour specimens from patients from the southwestern part of The Netherlands. Twenty-nine of 64 tumours showed mutations, of which 10 were non-missense mutations. The majority (9 of 10) of these non-missense mutations, including 7 nonsense mutations and 2 frameshift deletions, were null type mutations and could not be detected by immunohistochemical staining. Five of the 7 nonsense mutations were mutations at codon 213 (Arg-->Stop). The nature of the high prevalence of this nonsense mutation in our series of ovarian carcinomas remains unknown. In addition to the 9 null type mutations, a splice junction mutation was encountered. In conclusion, we have observed a high prevalence (13%) of ovarian tumours with null type mutations in exons 5-8 that did not result in immunostaining. Our data suggest that, especially in ovarian cancer, immunological assessment of TP53 is not an adequate tool to study TP53 alteration. A frequent nonsense mutation at codon 213 in 5 (8%) of 64 tumour specimens represents an important finding.

Ki-67 staining in benign, borderline, malignant primary and metastatic ovarian tumors: correlation with steroid receptors, epidermal-growth-factor receptor and cathepsin D.

Ki-67 immunoreactivity was studied in relation to immunohistochemically assessed expression of epidermal-growth-factor receptor (EGFR), estrogen receptor (ER) progesterone receptor (PgR) and cytosolic levels of cathepsin D in advanced human ovarian adenocarcinomas, borderline and benign cystadenomas and normal ovaries. A significantly higher number of Ki-67-positive cells were found in metastatic tumors vs. primary adenocarcinomas and in the total group of adenocarcinomas vs. benign/borderline cystadenomas. Cathepsin-D levels were also significantly higher in metastatic tumors than in primary adenocarcinomas, which in turn presented higher levels than were found in normal ovaries. However, no significant difference was observed between cathepsin-D levels in malignant adenocarcinomas and borderline/benign cystadenomas. Immunohistochemically assessed expression of ER and PgR was detected in variable percentages of epithelial tumor cells, and stromal cells were occasionally positive as well. In the group of primary adenocarcinomas, 46% were ER-positive and 34% were PgR-positive, although there was no significant difference between primary and metastatic lesions with respect to ER or PgR expression. Concordance between immunohistochemically assessed ER or PgR data and cytosolic ER and PgR levels measured with enzyme immunoassay was relatively low. EGFR, immunohistochemically assessed with MAb-EGFRI, was positive in 76% of the primary and in 78% of the metastatic adenocarcinomas. A strong positive association was detected between ER and PgR, and EGFR was observed to present a weak positive correlation with Ki-67 and ER. Cathepsin-D levels were not found to be significantly correlated with the expression of ER, PgR, EGFR or Ki-67.

Rectum obstruction in rats after local irradiation.

The dependence of radiation damage of the rectum of the rat on single and fractionated doses was studied. Results obtained were analyzed with the linear-quadratic model; an alpha/beta dose of 14 Gy could be derived.