Mechanisms of G1 checkpoint loss in resected early stage non-small cell lung cancer

Loss of the G1 checkpoint appears to be extremely common among virtually all neoplasms. A variety of genetic and epigenetic mechanisms have been demonstrated to play significant roles in this process. In a consecutive series of early stage non-small cell lung cancer (NSCLC), we have established the loss of expression of the G1 Cdk inhibitors p15INK4b) and p16INK4a by DNA methylation is very common (37%), and methylation of p16INK4a is tightly correlated with loss of expression of p16INK4a protein (P = 0.0018). Furthermore, methylation of p15INK4b and p16INK4a appear inversely correlated, although methylation of p15INK4b is an infrequent event in this cohort (4%). Methylation was detected in all stages of NSCLC equally, and did not correlate with survival in these patients. Evidence for methylation was more frequent in squamous cell carcinomas in comparison to other tumor histologies (P = 0.0156). In addition, over-expression of cyclin D1 was found to be tightly restricted (P = 0.0032) to those tumors that had retained wild-type expression of pRB, and did not correlate with methylation or expression of p16INK4a gene product. Although loss of p16INK4a function remains tightly correlated with pRB expression, loss of other regulatory elements in NSCLC such as p53 mutation and cyclin D1 over-expression appear independent of loss of the p16INK4a gene product.     

Multigene analysis of Rb pathway and apoptosis control in esophageal squamous cell carcinoma identifies patients with good prognosis

Deregulation of cell-cycle G(1)-restriction point control by disruption of Rb-pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin-dependent kinase inhibitor p16(INK4a) and the proapoptotic Bcl-2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G(1) restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16(INK4a), Rb, p21(CIP/WAF-1), p53, Bax and Bcl-2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP-PCR of exons 5-8. Loss of p16(INK4a), Rb, p21(CIP/WAF-1) or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl-2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21(CIP/WAF-1), p16(INK4a) and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.     

Prognostic implications of molecular and immunohistochemical profiles of the Rb and p53 cell cycle regulatory pathways in primary non-small cell lung carcinoma.

PURPOSE: Many studies have highlighted the aberrant expression and prognostic significance of individual proteins in either the Rb (particularly cyclin D1, p16INK4A, and pRb) or the p53 (p53 and p21Waf1) pathways in non-small cell lung cancer. We hypothesize that cumulative abnormalities within each and between these pathways would have significant prognostic potential regarding survival. EXPERIMENTAL DESIGN: Our study population consisted of 106 consecutive surgically resected cases of predominantly early-stage non-small cell lung cancer from the National Cancer Institute-Mayo Clinic series, and assessment of proteins involved both immunohistochemical (cyclin D1, p21Waf1, pRb, p16INK4A, and p53) and mutational analysis (p53) in relationship to staging and survival. RESULTS: Cyclin D1 overexpression was noted in 48% of the tumors, p16INK4A negative in 53%, pRb negative in 17%, p53 immunopositive in 50%, p53 mutation frequency in 48%, and p21(Waf1) overexpression in 47%, none with prognostic significance. Cyclin D1 overexpression in pRb-negative tumors revealed a significantly worse prognosis with a mean survival of 2.3 years (P = 0.004). A simultaneous p53 mutation dramatically reduced the mean survival time to 0.9 years (P = 0.007). Cyclin D1 overexpression with either a p53 mutation or a p53 overexpression was also associated with a significantly poorer prognosis (P = 0.0033 and 0.0063, respectively). CONCLUSIONS: Some cumulative abnormalities in the Rb and p53 pathways (e.g., cyclin D1 overexpression and p53 mutations) significantly cooperate to predict a poor prognosis; however, the complexity of the cell cycle protein interaction in any given tumor warrants caution in interpreting survival results when specific protein abnormalities are taken in isolation.     

Alterations of G1-S checkpoint in chordoma: the prognostic impact of p53 overexpression

BACKGROUND: To the authors' knowledge, little is known regarding the alterations of G(1)-S checkpoint and their significance in chordoma, a rare bone tumor. The authors investigated the clinicopathologic relevance of cell cycle abnormalities in chordoma. METHODS: The expression levels of p53, murine double minute 2 (MDM2), retinoblastoma protein (pRb), cyclin D1, p16(INK4a), and p27(Kip1) were investigated using immunohistochemical techniques; p53 mutations were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism, and mdm2 amplification was analyzed using real-time quantitative PCR. The results were compared with clinicopathologic parameters in 101 lesions. RESULTS: Approximately 10-45% of primary tumors presented alterations of p53, MDM2, cyclin D1, and pRb proteins; most tumors lacked expression of p16(INK4a) and p27(Kip1). Alterations of p53, MDM2, cyclin D1, and pRb proteins were found to have cooperative effects on both higher proliferative ability (MIB-1 labeling index [LI]) and increased nuclear pleomorphism, a previously described prognostic indicator for patients with chordoma. Multivariate analyses revealed that, among these alterations, p53 overexpression was the only independent factor for higher MIB-1 LI. At the genetic level, mdm2 gene amplification was detected in 15.4% of the lesions but did not correlate with MDM2 overexpression or other clinicopathologic parameters. No p53 mutations were detected in the current series. Survival analysis revealed that p53 overexpression, but no other cell cycle alterations, was associated with a reduced overall survival. CONCLUSIONS: Accumulation of cell cycle alterations led to an increased MIB-1 LI and nuclear pleomorphism, a previously described prognostic indicator in chordoma. The authors believe that p53 overexpression in particular is associated with an unfavorable prognosis in patients with chordoma.     

Alterations of the cyclin D1/pRb/p16(INK4A) pathway in multiple myeloma.

The retinoblastoma protein (pRb), p16(INK4A), D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G(1) regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5' CpG island hypermethylation of the p16(INK4A)gene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16(INK4A) was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fisher's test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16(INK4A) hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16(INK4A) hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16(INK4A) hypermethylation is frequent, inactivation of p16(INK4A) seems not to be involved in the pathogenesis of plasma cell disorders.     

Cyclin D1 expression in non-small-cell lung cancers: its association with altered p53 expression, cell proliferation and clinical outcome.

Cyclin D1, like p16INK4 (p16) and retinoblastoma (RB) proteins, participates in the cell cycle control at the G1-S transition. We have previously demonstrated altered p16 and RB protein status in non-small-cell lung cancers (NSCLCs) and their potential synergistic effect with altered p53 protein on proliferative activity (Kinoshita et al (1996) Cancer Res 56: 5557-5562). In the present study, cyclin D1 expression was studied by immunohistochemistry in the same cohort of 111 resected NSCLCs as in our previous study, and the amount of the cyclin D1 gene was analysed by Southern blot analysis in 29 NSCLCs. Cyclin D1 expression was analysed in relation to the status of p53, p16 and RB proteins, and proliferative activity determined by the Ki-67 index. It was also analysed in relation to survival of 77 patients with NSCLCs which were potentially curatively resected between 1990 and 1995. We found that: (1) cyclin D1 was expressed in 13 (11.7%) of 111 NSCLCs; (2) the cyclin D1 gene was neither significantly amplified nor rearranged; (3) cyclin D1 expression significantly correlated with altered p53 protein expression (P = 0.04), whereas it did not correlate with p16 and RB protein status; (4) proliferative activity tended to be higher in cyclin D1-positive (+) tumours than in cyclin D1-negative (-) tumours, although this difference was not statistically significant (P = 0.08); and (5) patients with cyclin D1+ tumours survived longer than patients with cyclin D1- tumours (5-year survival rates, 89% and 64% respectively, by the Kaplan-Meier method; P = 0.045 by the log-rank test), and cyclin D1 expression tended to be a favourable prognostic factor (P = 0.08 in univariate analysis). These findings suggest the involvement of cyclin D1 in the development and progression of NSCLCs, their proliferative activity and clinical outcome of NSCLC patients.     

Ovarian cancer cells that coexpress endogenous Rb and p16 are insensitive to overexpression of functional p16 protein

Defects of the 'Rb/cyclin D1/p16 pathway' have been shown to play a critical role in the development of virtually all human malignancies assessed. To determine the contribution of G1 phase cell cycle defects to ovarian tumorigenesis, we have examined a panel of normal and tumor ovarian tissues and ovarian cancer cell lines for the expression of Rb, p16 and cyclin D1 proteins. Unlike most types of human cancer whose development involves the loss of either Rb or p16 expression, we observed the coexpression of Rb, p16 and cyclin D1 in 82% of ovarian cancer tissues and cell lines. Furthermore, the growth and cell cycle distribution profiles of three ovarian cancer cell lines (ES-2, PA-1 and NIH OVCAR-3) that coexpressed Rb and p16, were found to be unaffected by adenoviral-mediated overexpression of functional p16 protein, indicating the existence of a defect(s) downstream from p16 in these cells. By contrast overexpression of ectopic p16 in the one ovarian cancer cell line (SK-OV-3) that expressed Rb but lacked p16 protein, resulted in a G1 growth arrest. These data suggest that defects of the 'Rb/cyclin D1/p16 pathway', other than the loss of Rb or p16, may play a major role in the development of ovarian cancer.     

Cyclin D1 overexpression in esophageal dysplasia: a possible biomarker for carcinogenesis of esophageal squamous cell carcinoma

There is controversy as to whether esophageal squamous dysplasia is a pre-cancerous lesion or a non-cancerous lesion. In this study, we conducted an immunohistochemical investigation of cyclin D1, retinoblastoma (Rb), p16INK4 and p27KIP1 expression in 36 squamous dysplasias and 34 early squamous cell carcinomas of the esophagus. The frequency of cyclin D1 overexpression was similar in dysplasias and early cancers (30% vs. 35%). Loss of p16INK4 and p27KIP1 expression was less frequent in dysplasias than in early cancers (p=0.005 and 0.001, respectively). Loss of Rb protein expression was not detected in dysplasia and rarely observed in early cancer (7%). The proliferation cell nuclear antigen index increased from moderate dysplasia to mucosal invasive carcinoma and was correlated significantly with the expression of cyclin D1, p16INK4 and p27KIP1 (p=0.0001, 0.003, and 0.007, respectively). Thus, this study found that cyclin D1 overexpression starts early in dysplasia and could be a useful marker for its malignant potentiality while reduction of p16INK4 and p27KIP1 occurs during the transformation from dysplasia to cancer. These findings suggest that esophageal dysplasia should be treated as a precancerous lesion.     

Loss of p16(INK4a) expression correlates with decreased survival in pediatric osteosarcomas

Abnormalities of the G1 cell-cycle checkpoint are commonly reported in cancers at various anatomic sites. pRB, p16(INK4a) and cyclin D1 are critical G1-checkpoint proteins responsible for maintaining the balance of cellular proliferation. We examined a series of 38 pediatric osteosarcomas for abnormal expression of pRB, p16(INK4a) and cyclin D1 by immunohistochemical analysis of archival biopsy specimens. Overall, 17/38 (45%) osteosarcomas showed evidence of G1-checkpoint abrogation, including 11/38 (29%) with loss of pRB expression and 6/38 (16%) with loss of p16(INK4a) expression. Cyclin D1 over-expression was not detected. There was an inverse correlation between loss of pRB and p16(INK4a) expression (p = 0.07). pRB and p16(INK4a) abnormalities were independent of site of disease, presence of metastasis at diagnosis and percentage of tumor necrosis in the resection specimen. Clinical follow-up was available on all patients (median 31.6 months, range 5.9-116 months). Absence of p16(INK4a) expression significantly correlated with decreased survival in univariate analysis (p = 0.03), while loss of pRB expression did not affect survival. Immunohistochemical analysis of p16(INK4a) expression in pediatric osteosarcomas may be a useful adjunctive marker of prognosis.     

Cyclin D1 and p16INK4A expression predict reduced survival in carcinoma of the anterior tongue.

Cyclin D1 and p16INK4A are molecules with pivotal roles in cell cycle control and the development of diverse human cancers, and overexpression of cyclin D1 and loss of p16INK4A expression are common genetic events in head and neck squamous cell carcinoma. The prognostic significance of these molecular events at different sites within the head and neck, however, remains controversial. Thus, we sought to determine the relationship between cyclin D1 and/or p16INK4A expression and disease outcome in squamous cell carcinoma of the anterior tongue. Immunohistochemical detection of nuclear proteins cyclin D1, p53, and p16INK4A, and the Ki-67 labeling index was undertaken in tissue sections from 148 tongue cancers treated by surgical resection. Nuclear antigen status was analyzed in relation to pathological variables, tumor recurrence, and patient survival. Statistical significance was assessed using chi2 analysis for pathological variables and the Kaplan-Meier method, log rank test, and the Cox proportional hazards model for survival parameters. Overexpression of cyclin D1 occurred in 68% of tumors (100 of 147) and was associated with increased lymph node stage (P = 0.014), increased tumor grade (P = 0.003), and reduced disease-free (P = 0.006) and overall (P = 0.01) survival. Loss of p16INK4A expression was demonstrated in 55% of tumors (78 of 143) and was associated with reduced disease-free (P = 0.007) and overall (P = 0.014) survival. Multivariate analysis confirmed that in addition to pathological stage and regional lymph node status, cyclin D1 overexpression and loss of p16INK4A expression are independent predictors of death from tongue cancer. Loss of p16INK4A in the presence of cyclin D1 overexpression conferred a significantly worse disease-free (P = 0.011) and overall (P = 0.002) survival at 5 years. p53 nuclear accumulation and the Ki-67 labeling index were not prognostic. These data indicate that cyclin D1 overexpression and loss of p16INK4A expression predict early relapse and reduced survival in squamous cell carcinoma of the anterior tongue. Simultaneous assessment of cyclin D1 and p16INK4A protein levels define subgroups of patients at increased risk of relapse and may be of clinical utility in optimizing therapy.     

p16INK4a loss and sensitivity in KSHV associated primary effusion lymphoma

The Kaposi's Sarcoma associated Herpes virus (KSHV) encodes two genes with the potential to affect the activity of the retinoblastoma protein (Rb). Open reading frame (orf) 72 encodes a D type cyclin (kcyc) that can elicit p16INK4a resistant cdk activity and orf73 encodes the latency associated nuclear antigen (LNA) that can bind Rb and neutralize E2F regulation. This indicates that, like papilloma and adenovirus associated malignancies, those associated with KSHV are defective with respect to their Rb pathway. To address this we investigated whether KSHV associated primary effusion lymphoma (PEL) derived cell lines are resistant to growth inhibition by p16INK4a. We provide evidence that ectopic expression of p16INK4a in these cells causes an Rb dependent G1 cell cycle block. Importantly, endogenous p16INK4a expression is not detected in six PEL derived cell lines and four primary PEL samples and examination of the p16INK4a locus shows deletion in two out of six and hypermethylation in four out of six PEL lines. Treatment of the latter with the demethylating agent 5'-aza-2' deoxycytidine leads to re-expression of p16INK4a protein. Taken together these results suggest that p16INK4a loss may be a cellular change frequently associated with PEL. They furthermore argue that despite the presence of KSHV DNA and expression of a latent gene program Rb function is intact in PEL.     

Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts

Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3-kinase and PI 3-kinase-related proteins (e.g. ATM), but it does not inhibit the activity of purified calmodulin-dependent protein kinase II (CaMKII). In the present study, we compared the effects of wortmannin and the CaMKII inhibitor KN62 on the response of normal human dermal fibroblast cultures to gamma radiation. We demonstrate that wortmannin confers a phenotype on normal fibroblasts remarkably similar to that characteristic of cells homozygous for the ATM mutation. Thus wortmannin-treated normal fibroblasts exhibit increased sensitivity to radiation-induced cell killing, lack of temporary block in transition from G1 to S phase following irradiation (i.e. impaired G1/S checkpoint), and radioresistant DNA synthesis (i.e. impaired S phase checkpoint). Wortmannin-treated cultures display a diminished capacity for radiation-induced up-regulation of p53 protein and expression of p21WAF1, a p53-regulated gene involved in cell cycle arrest at the G1/S border; the treated cultures also exhibit decreased capacity for enhancement of CaMKII activity post-irradiation, known to be necessary for triggering the S phase checkpoint. We further demonstrate that KN62 confers a radioresistant DNA synthesis phenotype on normal fibroblasts and moderately potentiates their sensitivity to killing by gamma rays, without modulating G1/S checkpoint, p53 up-regulation and p21WAF1 expression following radiation exposure. We conclude that CaMKII is involved in the radiation responsive signalling pathway mediating S phase checkpoint but not in the p53-dependent pathway controlling G1/S checkpoint, and that a wortmannin-sensitive kinase functions upstream in both pathways.     

Independent prognostic significance of cell cycle regulator proteins p16(INK4a) and pRb in advanced-stage ovarian carcinoma including optimally debulked patients: a translational research subprotocol of a randomised study of the Arbeitsgemeinschaft Gynaekologische Onkologie Ovarian Cancer Study Group

The purpose of the study is to test the hypothesis that expression of cell cycle regulatory proteins p16(INK4a) and pRb is significantly associated with prognosis in ovarian carcinomas. We performed immunohistochemical analysis of p16(INK4a) and pRb expression and correlated with survival in a series of 300 patients with FIGO stage IIb-IV ovarian carcinoma which were enrolled in a randomized prospective trial evaluating two different platinum and paxlitaxel chemotherapy combinations after radical surgery. p16(INK4a) negative tumours (17/300; 6%) had a significantly worse prognosis (univariate analysis, P<0.001; multivariate analysis: odds ratio 2.41, P=0.009). Among p16(INK4a)-positive tumours (283 out of 300; 94%), survival was better for patients with intermediate expression as compared to low or high expression levels (P=0.001). High expression levels of pRb were associated with an incremental deterioration of prognosis (univariate analysis, P=0.004; multivariate analysis: odds ratio 2.98, P=0.002). This observation held also true in the subgroup of optimally debulked patients (n=82), in whom the most important established prognostic factor, postoperative residual tumour cannot be applied. In conclusion p16(INK4a) and pRb are independent prognostic factors in advanced-stage ovarian carcinomas after radical surgery and postoperative chemotherapy. High pRb expression is a significant prognosticator in optimally debulked patients and may hold potential for subgroup stratification in postoperative treatment.     

苦参碱诱导人肝癌细胞分化、凋亡时对 G1细胞周期调节因子的调控

目的：观察苦参碱诱导人肝癌细胞分化、凋亡时,G1细胞周期调节因子的变化,探讨苦参碱对肿瘤细胞增殖的调控。方法：0、0．3、0．8、1．5g/L苦参碱作用HepG2细胞3天,免疫组化检测p53,Rb,p21,p27,p16,cyclinD1蛋白表达：原位杂交检测p53,cyclin D1mRNA的表达。真彩色图像分析对基因表达强弱进行定量。结果用Microsoft-Excel软件统计处理。结果：用药后细胞周期负调控因子p53,Rb,p21,p27,p16表达增强;正调控因子cyclinD1表达减弱。结论：苦参碱诱导人肝癌细胞HepG2分化、凋亡的机制可能与苦参碱上调了G1细胞周期负调节因子的表达,下调了G1期正调节因子cyclinD1表达有关。     

Inhibition of cell growth and induction of G1-phase cell cycle arrest in hepatoma cells by steroid extract from Meretrix meretrix

In this study, we report that the steroid extract 5alpha, 8alpha-epidioxycholest-6-ene-3beta-ol (MME) from Meretrix meretrix has the ability to inhibit growth of hepatoma cells and to induce G1-phase cell cycle arrest in two human hepatoma cell lines, HepG2 and Hep3B. HepG2 cells were more sensitive than Hep3B to MME. The extract markedly up-regulated the expression of p53 and p21WAF1/CIP1 in HepG2, suggesting that MME-induced G1 phase cell cycle arrest in HepG2 might be p53-dependent. Therefore, the up-regulation of p27KIP1and p16INK4A in both cell lines indicates that a p53-independent pathway might be involved in the mechanism of MME inducing cell cycle arrest. In conclusion, MME induces G1 phase cell cycle arrest via both p53-dependent and p53-independent pathways.     

Alterations of Rb, p16(INK4A) and cyclin D1 in the tumorigenesis of oral squamous cell carcinomas

Immunohistochemical analysis of Rb, p16(INK4A) and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16(INK4A) expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16(INK4A) was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16(INK4A) expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.