Aberrant Expression of pRb, p16, p14ARF, MDM2, p21 and p53 in Squamous Cell Carcinomas of Lung

Expression of cell cycle regulatory proteins in both the RB and p53 pathways was investigated in 50 cases of squamaous cell carcinoma (SCC) of the lung using immunohistochemical techniques.Abnormality of pRb and p16 expression was seen at the frequencies of 16% and 78%, respectively, and appeared to be in a reciprocal relationship. On the other hand, strong and diffuse p53 immunoreactivity was seen in 60% of SCCs. MDM2 and p14ARF expression. Moreover, strong p53 expression Moreover, strong p53 expression was not correlated with the expression of p21. In comparing clinicopathological status with immunohistochemical results, lack of p16 immunoreactivity was observed in the elderly group (over 65 years) as compared with the younger group (less than 65 years) Strong p53 expression was frequently observed in higher stages of SCC, with the developing tumor located in the central field of the lung. Similarly, the frequency of p14ARF expression was high in centrally developed SCC, but low in SCC developed in the periphery. These results suggest that disruption of the RB and p53 pathways is a frequent event in SCC, and that abnormal expression of p16 and p53 plays a more critical role than that of pRB, p14ARF and MDM2 in the development of SCC of the lung.     

p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2

The INK4a/ARF locus encodes two distinct tumor suppressors, p16INK4a and p14ARF. Although the contribution of p16INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14ARF lesions and their consequences. Recent data indicate that p14ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14ARF methylation. Taken together, these data support that epigenetic silencing of p14ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.     

Predictive significance of the alterations of p16INK4A, p14ARF, p53, and proliferating cell nuclear antigen expression in the progression of cervical cancer.

PURPOSE: The purpose of this research was to evaluate the clinical significance of p16INK4A, p14ARF, p53, and proliferating cell nuclear antigen (PCNA) expression in tumor progression of cervical cancer. DESIGN: Seventeen patients (40 samples) with consecutive cervical lesions from normal squamous epithelium, inflammation of the cervix to cervical intraepithelial neoplasm (CIN) and invasive cervical squamous cell cancer (SCC), or from CIN to SCC were collected for this study. Expression of p16INK4A, p14ARF, p53, and PCNA were detected by immunohistochemistry on paraffin-embedded sections. Human papillomavirus DNA was detected simultaneously with PCR and typed according to its DNA sequence. RESULTS: p16INK4A overexpression was significantly higher in CIN (75%) and in SCC (75%) than in normal or inflammation of the cervix (12.5%; P < 0.01, P < 0.05, respectively). The positive rate of p14ARF expression was higher in SCC (83%) than in normal/inflammation of the cervix (25%; P < 0.05). PCNA expression was negative in normal or inflammation of the cervix, but an increased in expression was seen in 63.2% in CIN and 100% in SCC (P < 0.01, P < 0.05). When the time interval for disease progression from initial biopsy to CIN 3 or invasive cancer was compared with states of p16INK4A expression, cases stained positive for p16INK4A progressed within 64.2 months as compared with 122.3 months among those stained negatively (P < 0.01). Cases with increased p14ARF expression also had a short time interval for disease progression of 78.8 months as compared with 108.3 months in cases that were p14ARF negative. Cases with stable or decreased p53 expression had the shortest time interval for progression of 32.3 months in contrast to cases with no p53 expression (113.9 months). However, cases with increasing p53 expression progressed within 60.8 months. CONCLUSIONS: Our results suggested that altered states of p16INK4A, p14ARF, p53, and PCNA may be valuable markers to predict the progression of cervical neoplasia.     

Deregulation of the TP53/p14ARF tumor suppressor pathway in low-grade diffuse astrocytomas and its influence on clinical course.

PURPOSE: The chromosome 9p21 region harbors three tumor suppressor genes, p14(ARF), p15(INK4b), and p16(INK4a), all of which can be targets for hypermethylation-associated inactivation in low-grade gliomas. p16(INK4a) and p15(INK4b) are critically involved in the RB1 pathway, whereas p14(ARF) acts as an upstream regulator of the TP53 pathway. The role of each tumor suppressor pathway in low-grade diffuse astrocytomas and their relationships with clinical behavior remain to be elucidated. EXPERIMENTAL DESIGN: We assessed the alterations of the RB1/CDK4/p16(INK4a)/p15(INK4b) and the TP53/MDM2/p14(ARF) pathways in 46 WHO grade II astrocytomas and analyzed their impact on prognosis. RESULTS: The TP53/MDM2/p14(ARF) pathway was altered in 32 of 46 cases (70%) by either TP53 mutation (25 cases) or p14(ARF) methylation (9 cases). The RB1/CDK4/p16(INK4a)/p15(INK4b) pathway was disrupted in 6 of 46 cases (13%) by either RB1 methylation (1 case), p16(INK4a) methylation (3 cases), or p15(INK4b) methylation or homozygous deletion (3 cases). Generally speaking, individual tumors thus tended to display alteration of only one component from both pathways. Any independently analyzed genetic alteration failed to provide statistically prognostic information. The alternate or simultaneous presence of TP53 mutation and p14(ARF) methylation emerged as a univariate predictor of a shorter progression-free survival (P = 0.0456) but was not statistically significant when age and extent of surgery were included in the analysis. CONCLUSIONS: Alternative disruption of the TP53/p14(ARF) pathway represents a frequent event in low-grade diffuse astrocytomas and correlates with an unfavorable clinical course. However, its value is unlikely to include prognostic utility that is independent of other conventional prognostic factors.     

Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma.

The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases, whereas p19ARF targets MDM2, a p53 inhibitory protein, for degradation. A deletion of CDKN2A would therefore disturb both pathways. To explore the p53 pathway genes as a functional unit in diffuse large B cell non-Hodgkin's lymphomas (DLCL), we wanted to see whether there exists mutually exclusiveness of aberrations of CDKN2A, MDM2 and p53, since this has not been analysed previously. We investigated 37 DLCL for aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigenetic, genetic and/or protein levels. Homozygous deletion of CDKN2A was detected in seven (19%) of 37 tumours, and another three cases were hypermethylated at the 5' CpG island of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue for p16 confirmed these results, as all tumours with alterations of CDKN2A were p16 immunonegative. We found p53 mutations in eight (22%) cases and MDM2 overexpression in 16 (43%) tumours. Twenty-three (62%) tumours had alterations of one or more p53 pathway components (p53, p19ARF and MDM2). Furthermore, 7/9 (78%) p16-immunonegative tumours showed co-aberration of p53 and/or MDM2. The lack of correlation between these aberrations suggests that DLCL acquire additional growth advantage by inactivating both of these critical regulatory pathways.     

Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C,hepatitis B and alcoholic liver cirrhosis

Major etiologic factors associated with human hepatocellular carcinomas (HCCs) include infection with hepatitis C (HCV) and hepatitis B virus (HBV), excess alcohol intake and aflatoxin B(1) exposure. While the G-->T p53 mutation at codon 249 has been identified as a genetic hallmark of HCC caused by aflatoxin B(1), the genetic profile associated with other etiologic factors appears to be less distinctive. In our study, we screened HCCs resulting from HCV infection (51 cases), HBV infection (26 cases) or excess alcohol intake (23 cases) for alterations in genes involved in the RB1 pathway (p16(INK4a), p15(INK4b), RB1, CDK4 and cyclin D1), the p53 pathway (p53, p14(ARF) and MDM2) and the Wnt pathway (beta-catenin, APC). Alterations of the RB1 pathway, mainly p16(INK4a) methylation, loss of RB1 expression and cyclin D1 amplification, were most common (69-100% of cases). There was a significant correlation between loss of RB1 expression and RB1 methylation. All 24 HCCs with RB1 promoter methylation lacked RB1 expression, while none of the 67 cases with RB1 expression exhibited RB1 methylation (p < 0.0001), suggesting that promoter methylation is a major mechanism of loss of RB1 expression in HCCs. Alterations of the p53 pathway consisted mostly of p53 mutations or p14(ARF) promoter methylation (20-48%). Mutations of the p53 gene were found at a similar frequency (13-15%) in all etiologic groups, without any consistent base change or hot spot. Mutations of beta-catenin were found in 13-31% of cases, while no APC mutations were detected in any of the HCCs analyzed. With the exception of only 3 of 39 cases (8%), cyclin D1 amplification and beta-catenin mutations were mutually exclusive, supporting the view that cyclin D1 is a target of the Wnt signaling pathway. Overall, the RB1, p53 and Wnt pathways were commonly affected in HCCs of different etiology, probably reflecting common pathogenetic mechanisms, i.e., chronic liver injury and cirrhosis, but tumors associated with alcoholism had more frequent alterations in the RB1 and p53 pathways than those caused by HCV infection.     

Frequent alterations of the p14(ARF) and p16(INK4a) genes in primary central nervous system lymphomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in the tumorigenesis of primary central nervous system lymphomas (PCNSLs), we have analyzed p14(ARF), p16(INK4a), RB1, p21(Waf1), and p27(Kip1) status in a series of their 18 sporadic cases of diffuse large B-cell lymphoma, using methylation-specific PCR, differential PCR, and immunohistochemistry. Homozygous deletion or methylation of p14(ARF) was detected in 10 (56%) PCNSLs, and they were almost entirely deletions (except 1 case). A total of 11 (61%) PCNSLs demonstrated homozygous deletion (6 cases) or methylation (5 cases) of p16(INK4a). Six tumors showed both p14(ARF) and p16(INK4a) homozygous deletions. Hypermethylation of the RB1 and the p27(Kip1) promoter region was detected in 2 (11%) cases, whereas p21(Waf1) methylation was not detected in any. Immunohistochemistry revealed loss of p14(ARF) and p16(INK4a) expression in 10 (56%) samples, correlating with the gene status. Four cases showed independent negative immunoreactivity for pRB and p27(Kip1), and nearly one-half of cases (8 of 18; 44%) were characterized by lack of p21(Waf1) expression. These results indicate that inactivation of p14(ARF) and p16(INK4a) by either homozygous deletion or promoter hypermethylation represents an important molecular pathogenesis in PCNSLs. Hypermethylation of RB1, p21(Waf1), and p27(Kip1) appears to be of minor significance, these genes being independently methylated in PCNSLs.     

Ratio of expression of p16INK4a to p14ARF correlates with the progression of non-small cell lung cancer.

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16(INK4a) and p14(ARF), through the use of independent first exons and shared exons 2 and 3. p16(INK4a) is a cyclin-dependent kinase inhibitor, whereas p14(ARF) regulates the cell cycle through a p53 and MDM2-dependent pathway. We have examined the expression of p16(INK4a) and p14(ARF) using competitive RT-PCR in 60 non-small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16(INK4a) and p14(ARF) were nearly equal or the intensity of the p16(INK4a) band slightly exceeded that of p14(ARF) in the normal lung tissues (n = 60). In 38 tumors the intensity of the p16(INK4a) band was similar to or slightly weaker than that of p14(ARF). In 6 tumors the intensity of the p16(INK4a) band was weaker than that of p14(ARF). In 15 tumors the intensity of the p14(ARF) band was very strong and the p16(INK4a) band was barely visible. In only one tumor was the intensity of the p16(INK4a) band very strong, while the band of p14(ARF) was barely visible. The ratio of the intensity of p16(INK4a) to p14(ARF) had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II - IV tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the p stage I tumors (P = 0.036). The T2 - 4 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the T1 tumors (P = 0.005). The N1 - 3 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the N0 tumors (P = 0.014). Our results suggest that the ratio of expression of p16(INK4a) to p14(ARF) tends to decrease during the progression of NSCLC.     

Dysfunction of p53 pathway in human colorectal cancer: analysis of p53 gene mutation and the expression of the p53-associated factors p14ARF, p33ING1, p21WAF1 and MDM2

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).     

Alterations of p14ARF, p53, and p73 genes involved in the E2F-1-mediated apoptotic pathways in non-small cell lung carcinoma.

Overexpression of E2F-1 induces apoptosis by both a p14ARF-p53- and a p73-mediated pathway. p14ARF is the alternate tumor suppressor product of the INK4a/ARF locus that is inactivated frequently in lung carcinogenesis. Because p14ARF stabilizes p53, it has been proposed that the loss of p14ARF is functionally equivalent to a p53 mutation. We have tested this hypothesis by examining the genomic status of the unique exon 1beta of p14ARF in 53 human cell lines and 86 primary non-small cell lung carcinomas and correlated this with previously characterized alterations of p53. Homozygous deletions of p14ARF were detected in 12 of 53 (23%) cell lines and 16 of 86 (19%) primary tumors. A single cell line, but no primary tumors, harbored an intragenic mutation. The deletion of p14ARF was inversely correlated with the loss of p53 in the majority of cell lines (P = 0.02), but this relationship was not maintained among primary tumors (P = 0.5). E2F-1 can also induce p73 via a p53-independent apoptotic pathway. Although we did not observe inactivation of p73 by either mutation or DNA methylation, haploinsufficiency of p73 correlated positively with either p14ARF or p53 mutation or both (P = 0.01) in primary non-small cell lung carcinomas. These data are consistent with the current model of p14ARF and p53 interaction as a complex network rather than a simple linear pathway and indicate a possible role for an E2F-1-mediated failsafe, p53-independent, apoptotic pathway involving p73 in human lung carcinogenesis.     

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.     

Correlation of abnormal RB, p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormalities, and clinical features in 103 primary non-small cell lung cancers.

This study was performed to determine the frequency of inactivation and clinical correlates in non-small cell lung cancer (NSCLC) of three known tumor suppressor genes [TSGs; RB, MTS1/CDKN2 (p16), and p53] and various regions of 3p loss of heterozygosity (LOH) as other major potential TSG sites. Paraffin sections from 103 resected NSCLCs were analyzed for expression of pRB, p16, and p53 by immunohistochemistry, whereas DNA from tumor and normal tissue were tested for LOH at 3p25-26, 3p21, and 3p14. Previously published LOH data for 5q, 11p, 17q, and 18q were also available. Loss of pRB or p16 expression and overexpression of p53 were considered abnormal. The immunohistochemical and LOH data were correlated with a variety of clinical parameters including stage, age, sex, smoking history, and survival. With respect to pRB, p16, and p53, the tumors could be grouped into four categories: normal for all three proteins (21%); abnormal for pRB or p16 and normal for p53 (30%); normal for pRB and p16 and abnormal for p53 (20%); and abnormal in both pathways (28%). Aberrant expression of pRB, p16, p53, and 3p LOH, either individually or in combination, was not associated with survival differences or any other clinical parameters, with the exception that pRB/pl6 abnormalities were more common in older patients (P = 0.0005). pRB and p16 expression showed a strong inverse correlation (P = 0.002), whereas there was no correlation between expression of pRB, p16, and p53. Abnormal expression of any of the three genes inversely correlated with K-ras codon 12 mutations (P = 0.004), but not with 3p LOH or LOH at other TSG loci. We conclude that resectable NSCLCs show distinct patterns of TSG inactivation, but that no clear clinical correlates exist either alone or in combination for pRB, p16, p53, and 3p abnormalities.     

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.     

Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for human astrocytic gliomas with G1-S transition control gene abnormalities

Deregulation of G1-S transition control in cell cycle is one of the important mechanisms in the development of human tumors including astrocytic gliomas. We have previously reported that approximately two-thirds of glioblastomas (GBs) had abnormalities of G1-S transition control either by mutation/homozygous deletion of RB1 or CDKN2A p16INK4A), or amplification of CDK4 (K. Ichimura et al., Oncogene, 13: 1065-1072, 1996). However, abnormalities of G1-S transition control genes may induce p53-dependent apoptosis in cells. Recent investigations suggest that p14ARF is induced in response to abnormal cell cycle entry and results in p53 accumulation by inhibiting MDM2-mediated transactivational silencing and degradation of p53. To investigate the roles of the G1-S transition control system and the p14ARF/MDM2/p53 pathway in the development of astrocytic gliomas, we examined abnormalities of genes involved in these regulatory pathways in a total of 190 primary human astrocytic gliomas of different malignancy grades [136 GBs, 39 anaplastic astrocytomas (AAs) and 15 astrocytomas (As)]. Sixty-seven percent of GBs (91/136) and 21% of AAs (8/39) had abnormalities of the G1-S control system either by mutation/homozygous deletion of RB1, CDKN2A or CDKN2B, or amplification of CDK4. Seventy-six percent of GBs (103 of 136), 72% of AAs (28 of 39), and 67% of As (10 of 15) had deregulated p53 pathway either by mutation of TP53, amplification of MDM2, or homozygous deletion/mutation of p14ARF. When all of the data were combined and compared, 96% of GBs (87 of 91) and 88% of AAs (7 of 8) with abnormal G1-S transition control also had deregulated p53 pathway. Thus, we demonstrate that deregulation of the G1-S transition control system was almost always accompanied by inactivation of the p53 pathway, clearly illustrating the cooperative roles of these two systems in the development/progression of primary human astrocytic gliomas.     

CIN和子宫颈鳞癌的HPV16/18感染与p53、MDM2蛋白表达的关系

目的研究子宫颈鳞癌及C IN的HPV感染、p53蛋白和M DM 2蛋白的表达及三者之间的相互关系。方法选取子宫颈鳞癌61例、C IN 47例、正常子宫颈组织54例,分别采用原位杂交技术检测HPV 16/18感染,用免疫组化技术S-P法检测p53蛋白和M DM 2蛋白。结果HPV 16/18感染率鳞癌组为62.3%(38/61),C IN组为70.2%(33/47),对照组为16.7%(9/54)。C IN组和鳞癌组的HPV 16/18的感染率均显著高于对照组(P值均<0.05)。而C IN组与鳞癌组的HPV 16/18感染率差异无显著性(P>0.05)。C IN组、鳞癌组、对照组的p53阳性率分别为19.1%(9/47)、26.2%(16/61)、3.7%(2/54)。C IN组、鳞癌组、对照组的M DM 2阳性率分别为14.9%(7/47)、26.2%(16/61)、7.4%(4/54)。C IN组和鳞癌组的p53阳性率均显著高于对照组(P值均<0.05),C IN组与鳞癌组比较差异无显著性(P>0.05)。M DM 2阳性率鳞癌组显著高于对照组(P<0.05),而在C IN组和鳞癌组之间、C IN组和对照组之间差异均无显著性(P值均>0.05)。在本实验各组中,p53与M DM 2之间的阳性率均无统计学相关(P值均>0.05)。p53阳性率和HPV 16/18感染之间差异无显著性(P值均>0.05),而M DM 2的阳性率与HPV 16/18感染仅在子宫颈鳞癌组呈正相关(P<0.05)。结论C IN及子宫颈鳞癌与HPV 16/18感染关系密切;p53和M DM 2均参与子宫颈癌及癌前病变的发生,但p53的突变、M DM 2的扩增和过表达均不占主导地位。     

Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer

Inactivation of the P16 (INK4A)/retinoblastoma (RB) or TP53 biochemical pathway is frequent event in most human cancers. Recent evidence has shown that P14ARF binds to MDM2 leading to an increased availability of wild type TP53 protein. Functional studies also support a putative tumor suppressor gene function for p14ARF suggesting that p14ARF or p53 inactivation may be functionally equivalent in tumorigenesis. To study the relative contribution of each pathway in tumorigenesis, we analysed and compared alterations of the p16, p14ARF and p53 genes in 38 primary non-small cell lung cancers (NSCLCs) (19 adenocarcinomas and 19 squamous carcinoma). The p16 tumor suppressor gene was inactivated in 22 of 38 (58%) tumors. Twelve of these samples (31%) had homozygous deletions by microsatellite analysis; eight of them (21%) had p16 promoter hypermethylation detected by Methylation Specific PCR (MSP) and the remaining two (5%) harbored a point mutation in exon 2 by sequence analysis. The absence of P16 protein in every case was confirmed by immunohistochemistry. Fourteen of the 22 tumors with p16 inactivation also inactivated the p14ARF gene (12 with homozygous deletions extending into INK4a/ARF and two with exon 2 mutations). Mutations of p53 were found in 18 (47%) of the tumors and nine of them (50%) harbored p14ARF inactivation. Thus, an inverse correlation was not found between p14ARF and p53 genetic alterations (P=0.18; Fisher Exact Test). Our data confirm that the p16 gene is frequently inactivated in NSCLC. Assuming that 9p deletion occurs first, the common occurrence of p53 and p14ARF alterations suggests that p14ARF inactivation is not functionally equivalent to abrogation of the TP53 pathway by p53 mutation.