Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal-type, and 20 of diffuse-type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO-III, and YCC-2, -3, -16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki-67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal-type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27(KIP1) expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21(WAF1) and low-grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor-suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low-grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials.     

Immunohistochemical expression of retinoblastoma gene product (Rb), p53 protein, MDM2, c-erbB-2, HLA-DR and proliferation indices in human urinary bladder carcinoma

Archival biopsy specimens from transitional cell bladder cancers (n=88) were analysed immunohistochemically for the expression of the retinoblastoma (Rb) gene protein, p53, mdm2, c-erbB-2, HLA-DR antigen and proliferation indices. An altered nuclear expression of Rb, p53 and mdm2 was observed in 55.2%, 33.3% and 18.2% of tumors respectively. Cytoplasmic membrane immunoreactivity (>25% tumor cells) for c-erbB-2 was detected in 14.1% of tumors and aberrant HLA-DR antigen cytoplasmic staining (>5% of tumor cells) in 22.2% of the cases. P53 overexpression was associated with higher tumor grade and stage. Aberrant HLA-DR antigen expression and PCNA were also correlated with the grade of differentiation and tumor stage. MIB1 was statistically correlated with stage. pRb scores and HLA-DR antigen expression were correlated with proliferation activity as determined by PCNA and MIB1 immunostaining. p53 protein was also strongly correlated with the proliferation index PCNA. A strong correlation between PCNA and MIB1 (p<0.0001) was also found. In addition a statistically positive correlation between p53 and HLA-DR antigen expression was observed. Our data show that, although pRb and p53 protein expressions are not associated between them, they may contribute to the growth fraction of the bladder cancer. In addition, p53 and HLA-DR antigen expression could be indicators of aggressive behavior of bladder cancer.     

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs)

BACKGROUND: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors. MATERIAL AND METHODS: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods. RESULTS: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055). CONCLUSIONS: Our findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.     

Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas

BACKGROUND: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells. MATERIAL AND METHODS: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods. RESULTS: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066). CONCLUSIONS: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.     

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.     

Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) expression in the successive steps of cervical neoplasia and to determine its correlation with angiogenesis and p53 status. STUDY DESIGN: Immunohistochemical staining with a VEGF monoclonal antibody was performed on a total of 161 cervical specimens representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 30 CIN 3 and 86 squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody to CD34. Computerized image analysis was used to evaluate microvessel density (MVD). p53 Status was determined by immunohistochemistry and direct sequencing of exons 5-8 of the p53 gene. RESULTS: VEGF expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < .05). MVD increased significantly with cervical neoplasia progression, from normal epithelium, through CIN, to squamous cell carcinoma (P < .001). A strong correlation was observed between VEGF expression and MVD (P < .001). p53 Protein expression was not detected in the normal epithelium or in CIN 1, while 3 (10%) of 30 CIN 3 and 28 (33%) of 86 squamous cell carcinomas were positive for p53. VEGF expression correlated statistically with p53 protein expression (P < .001). In double VEGF- and p53-stained sections, the 2 markers were generally expressed in the same tumor cells. Of the 4 p53 gene mutations, 3 exhibited strong VEGF expression, and 1 exhibited moderate VEGF expression. VEGF expression did not correlate significantly with outcome variables in patients with squamous cell carcinoma. CONCLUSION: Our results suggest that VEGF expression is involved in the promotion of angiogenesis in cervical neoplasia and that p53 is likely to be involved in the regulation of VEGF expression.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

p53 and the malignant progression of Barrett's esophagus

Barrett's esophagus (BE) is a metaplastic disorder in which specialized columnar epithelium replaces healthy squamous epithelium (intestinal metaplasia). Even though its pathophysiology and the steps of its neoplastic progression are not completely understood, BE can be considered as a complication of gastroesophageal reflux disease (GERD). Given that esophageal adenocarcinoma, which is continually increasing in the Western world, still has a poor prognosis and suffers from late diagnosis, and because BE is a precancerous lesion, there is a strong need for good molecular markers of malignant progression in Barrett's metaplasia (BM). The aim of this review is to examine the published data regarding the role that assessment of p53 may play in the management of BE, trying to understand if it may be a useful marker to early diagnose BE malignant transformation.     

Bile salt exposure increases proliferation through p38 and ERK MAPK pathways in a non-neoplastic Barrett's cell line

Bile reflux has been implicated in the neoplastic progression of Barrett's esophagus (BE). Bile salts increase proliferation in a Barrett's-associated adenocarcinoma cell line (SEG-1 cells) by activating ERK and p38 MAPK pathways. However, it is not clear that these findings in cancer cells are applicable to non-neoplastic cells of benign BE. We examined the effect of bile salts on three human cell lines: normal esophageal squamous (NES) cells, non-neoplastic Barrett's cells (BAR cells), and SEG-1 cells. We hypothesized that bile salt exposure activates proproliferative and antiapoptotic pathways to promote increased growth in BE. NES, BAR, and SEG-1 cells were exposed to glycochenodeoxycholic acid (GCDA) at a neutral pH for 5 min. Proliferation was measured by Coulter counter cell counts and a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. GCDA-induced MAPK activation was examined by Western blot analysis for phosphorylated ERK and p38. Apoptosis was measured by TdT-mediated dUTP nick-end labeling and annexin V staining after GCDA and UV-B exposure. Statistical significance was determined by ANOVA. NES cells exposed to 5 min of GCDA did not increase cell number. In BAR cells, GCDA exposure increased cell number by 31%, increased phosphorylated p38 and ERK levels by two- to three-fold, increased BrdU incorporation by 30%, and decreased UV-induced apoptosis by 15-20%. In conclusion, in a non-neoplastic Barrett's cell line, GCDA exposure induces proliferation by activation of both ERK and p38 MAPK pathways. These findings suggest a potential mechanism whereby bile reflux may facilitate the neoplastic progression of BE.     

EZH2 Is Associated with Malignant Behavior in Pancreatic IPMN via p27Kip1 Downregulation

Background

The epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression.

Methods

Fifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27^Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of EZH2 and p27^Kip1 mRNA were analyzed by real time PCR.

Results

In immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma). EZH2 expression displayed a similar pattern (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma) with cell proliferative activity. EZH2 expression in malignant (CIS and invasive carcinoma) IPMNs was significantly higher than that in adenoma and borderline-atypia IPMNs. EZH2 expression level in IPMN lesions was positively correlated with the Ki-67 positive nuclear ratio (p<0.0001). EZH2-positive cells in malignant IPMN did not express p27^Kip1. EZH2 mRNA expressions in malignant lesions were significantly higher than those in benign lesions (p<0.0001). In contrast, p27^Kip1 mRNA in malignant lesions was significantly decreased compared to those in benign lesion (p<0.05), and there was an inverse correlation between EZH2 and p27^Kip1 mRNA levels (p = 0.0109).

Conclusion

EZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27^Kip1.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

p27kip1 overexpression promotes paclitaxel-induced apoptosis in pRb-defective SaOs-2 cells

p27kip1 is a cyclin-dependent kinase (CDK) inhibitor, which controls several cellular processes in strict collaboration with pRb. We evaluated the role of p27kip1 in paclitaxel-induced apoptosis in the pRb-defective SaOs-2 cells. Following 48 h of exposure of SaOs-2 cells to 100 nM paclitaxel, we observed an increase in p27kip1 expression caused by the decrease of the ubiquitin-proteasome activity. Such increase was not observed in SaOs-2 cells treated with the caspase inhibitors Z-VAD-FMK, suggesting that p27kip1 enhancement at 48 h is strictly related to apoptosis. Finally, we demonstrated that SaOs-2 cells transiently overexpressing the p27kip1 protein are more susceptible to paclitaxel-induced apoptosis than SaOs-2 cells transiently transfected with the empty vector. Indeed, after 48 h of paclitaxel treatment, 41.8% of SaOs-2 cells transiently transfected with a pcDNA3-p27kip1 construct were Annexin V-positive compared to 30.6% of SaOs-2 cells transfected with the empty vector (P < 0.05). In conclusion, we demonstrated that transfection of the pRb-defective SaOs-2 cells with the p27kip1 gene via plasmid increases their susceptibility to paclitaxel-induced apoptosis. The promoting effect of p27kip1 overexpression on apoptosis makes p27kip1 and proteasomal inhibitors interesting tools for therapy in patients with pRb-defective cancers.     

All trans-retinoic acid induces apoptosis via p38 and caspase pathways in metaplastic Barrett's cells

Retinoids such as all trans-retinoic acid (ATRA) have been used as chemopreventive agents for a number of premalignant conditions. To explore a potential role for retinoids as chemopreventive agents for Barrett's esophagus, we studied ATRA's effects on apoptosis in a nonneoplastic, telomerase-immortalized, metaplastic Barrett's cell line. We treated the Barrett's cells with ATRA in the presence and absence of inhibitors to p53 (pSRZ-siRNA-p53), p38 (SB-203580 and p38 siRNA), and the caspase cascade (z-Val-Ala-Asp-fluoromethyl ketone). We determined the effects of ATRA and the various inhibitors on apoptosis using cell morphology, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining, cleaved caspase-3 immunofluorescence, and Annexin V staining. We also determined how ATRA in the presence and absence of the inhibitors affected apoptosis following low-dose UV-B irradiation. ATRA induced apoptosis and increased the expression of p53 protein in a dose-dependent fashion. The apoptotic effect of ATRA was abolished by treatment with inhibitors of both p38 and caspase, but not by p53 interfering RNA (RNAi). Inhibition of p38 also prevented expression of cleaved caspase-3, suggesting that ATRA activates p38 upstream of the caspase cascade. We found that ATRA sensitized immortalized Barrett's cells to apoptosis induced by low-dose UV-B irradiation via a similar mechanism. ATRA induces apoptosis in Barrett's epithelial cells and sensitizes them to apoptosis induced by UV-B irradiation via activation of p38 and the caspase cascade, but not through p53. This study elucidates molecular pathways whereby retinoid treatment might prevent carcinogenesis in Barrett's metaplasia and suggests a potential role for the use of safer retinoids for chemoprevention in Barrett's esophagus.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.Key words: breast cancer, senescence, retinoblastoma, p130, p107     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

Cross‐talk between T‐Ag presence and pRb family and p53/p73 signaling in mouse and human medulloblastoma

The formation and progression of mudulloblastoma (MB) is poorly understood. However, somatic inactivation of pRb/p105, in combination with a somatic or a germ‐line TP53 inactivation, leads to MB in a mouse model. Presently, there is no specific evidence of pathway/s alterations for the other two members of the retinoblastoma family, pRb2/p130 and/or p107 in MB. JC virus (JCV) is a human polyomavirus. Although there is no firm evidence that this virus plays a causal role in human neoplasia, it has been clearly proven that JCV is highly oncogenic when injected into the brain of experimental animals. The mechanism of JCV‐induced tumorigenesis is not entirely clear. However, several studies relate the oncogenic properties of JCV mainly to its early protein large T‐antigen (T‐Ag), which is able to bind and inactivate both TP53 and Rb family proteins. Here, we compared the protein expression profiles of p53, p73, pRb family proteins, and PCNA, as main regulators of cell proliferation and death, in different cell lines of mouse primitive neuroectodermal tumors (PNET), either T‐Ag‐positive or ‐negative, and in human MB cell lines. Our goal was to determine if changes in the relative expression of these regulators could trigger molecular perturbations underlying MB pathogenesis in mouse and human cells. Our results support that the presence of JCV T‐Ag may interfere with the expression of pRb family proteins, specific p73 isoforms, and p53. In turn, this “perturbation” may trigger a network of signals strictly connected with survival and apoptosis. J. Cell. Biochem. 110: 182–190, 2010. © 2010 Wiley‐Liss, Inc.     

ARHI和P130 在子宫内膜癌中的表达和意义

目的：探讨ARHI和P130 蛋白在子宫内膜癌中的表达状况,同时分析两因子在子宫内膜癌中的关联性。方法：收集80 例子 宫内膜癌,子宫内膜非典型增生型80 例及同时期因子宫肌瘤切除的40 例正常的子宫内膜的详细病例及高质量切片,设计分组 并使用免疫组化S-P法检测ARHI 蛋白和pRb2/P130的表达状况,并探究此两因子与子宫内膜癌的关联性。结果：ARHI蛋白阳 性表达率在正常子宫内膜组（97.50%）,子宫内膜非典型增生型组（53.75%）和子宫内膜癌组中（38.75%）三组中依次递减,组间差 异有统计学意义,P<0.05。ARHI蛋白阳性表达降低或缺失与子宫内膜癌的的恶性程度,手术病理分期有关(P<0.05),与子宫内膜 癌的类型无关（P>0.05）。Rb2/p130 蛋白的阳性表达率在三组中逐渐降低,在正常子宫内膜组（100%）,子宫内膜非典型增生型组 （56.25 %）及子宫内膜癌组（36.25 %）,组间差异有统计学意义,P<0.05。Rb2/P130 的阳性表达率降低或缺失与子宫内膜癌的组织 恶性程度,手术病理级别和组织学类型有关(P<0.05),同时Spearman 等级相关性分析表明：ARHI蛋白和Rb2/p130 蛋白表达在子 宫内膜癌中为正相关,r=0.435。结论：ARHI蛋白和Rb2/p130 的阳性表达率降低或缺失可能在某种程度上导致子宫内膜癌的产生 和恶化。