Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

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.     

Methylation Status of P16Ink4a in Human Papillomavirus-Associated Cancer of Oral Cavity and Oropharynx in Northeastern Thailand

Background: Over-expression of p16INK4a protein is a biomarker for human papillomavirus (HPV)-associated cervical cancer. However, absence of p16INK4a protein expression in HPV-associated cancer of the oral cavity and oropharynx has been reported. Among a number of possible reasons for this is methylation, which is frequently noted in the promoter region of p16INK4a and is associated with silencing of the gene and disease severity. Methods: We investigated the relationships between p16INK4a protein expression, HPV infection and methylation status of the p16INK4a promoter in cancers of the oral cavity and oropharynx. Fifty-three formalin-fixed paraffin-embedded (FFPE) cancer tissue samples from the oral cavity (49 cases) and oropharynx (4 cases) were studied. P16INK4a protein expression was determined using immunohistochemical staining (IHC). Additional oral tissues lacking squamous intraepithelial lesions (SILs), and cervical tissues with high-level SILs, were used as negative and positive controls, respectively. High-risk HPV infection was detected using HPV E6/E7 mRNA in situ hybridization. Methylation status of the p16INK4a promoter was investigated using sodium bisulfite treatment and methylation-specific PCR (MS-PCR).Results: HPV infection was found in 40.8% (20/49) and 50.0% (2/4) of oral cavity and oropharynx cancers, respectively. Promoter methylation of p16INK4a occurred in 73.6 % of all cases and differed significantly in frequency between HPV-positive (90.9%, 20/22) and HPV-negative (61.3%, 19/31) samples. Expression of p16INK4a was found in 35.8% (19/53) and commonly detected in samples with p16INK4a unmethylation (79.5%). Interestingly, the silencing of p16INK4a (64.2%, 34/53) was significantly associated with methylation status (91.2%, 31/34), especially in HPV-infected samples in which the p16INK4a promoter was methylated (52.9%, 18/34). Conclusions: This result demonstrated high frequency of p16INK4a promoter methylation status in HPV-associated HNSCC subsets that could influence the silent p16INK4a expression and might promote disease severity.     

Relationship among human papillomavirus infection, p16(INK4a), p53 and NF-κB activation in penile cancer from northern Thailand

Human papillomavirus (HPV) E6 and E7 oncoproteins are essential factors for HPV oncogenesis. These E6 and E7 gene products play a central role in the induction of malignant transformation by interacting with several cellular regulatory proteins such as p16(INK4a), p53 and nuclear factor κB (NF-κB). In the present study, conducted in northern Thailand, HPV-DNA was detected in penile cancer cases using an in situ hybridization procedure and p16(INK4a), p53 and NF-κB were detected by immunohistochemistry. Using the cell cycle regulatory proteins p16(INK4a) (61.5%) and p53 (71.8%), it was found that of the 51 cases, 39 (76.5%) were HPV-DNA-positive in penile cancer. On the other hand, 25% p16(INK4a) and 75% p53, respectively, were found in HPV-negative cases. Prevalence of HPV infection (76.5%) was shown in penile cancer cases in northern Thailand. No difference was found between HPV-positive and HPV-negative cases with respect to the presence of the cell cycle regulatory protein p53. On the other hand, p16(INK4a) was found to be different between HPV-positive and HPV-negative cases. Inactivation of tumor suppressor genes, such as p16(INK4a) and p53, to genetic instability, cell immortalization, accumulation of mutations and cancer formation, with or without HPV and irrespective of HPV infection, is therefore suggested. Of the 39 HPV-positive cases, 35 (89.7%) were NF-κB-positive in the nucleus, 29 (74.4%) in the cytoplasm and 37 (94.9%) in the nucleus and/or cytoplasm. NF-κB was detected in 4 (33.3%) of the 12 HPV-negative cases. Therefore, we propose that penile cancer cases with HPV infection are more likely to activate NF-κB than those without HPV infection.     

Nuclear co-expression of p14ARF and p16INK4A in uterine cervical cancer-derived cell lines containing HPV

The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.     

p73 is over-expressed in vulval cancer principally as the Delta 2 isoform.

p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expression in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 Delta 2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with hypermethylation or mutation of p16(INK4a) in HPV-negative cases. There was a close correlation between expression of p73 and p14(ARF) in cancers with loss of p53 function. The frequent over-expression of p73 Delta 2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process.     

p53 mutations in cervical carcinogenesis--low frequency and lack of correlation with human papillomavirus status.

p53 gene aberrations are common in human malignancies, and recent studies suggest that in cervical carcinoma p53 function is inactivated either by complex formation with human papillomavirus (HPV) E6 product or by gene mutation. Using polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE), we examined the mutational status of the four ''hotspot'' regions of the p53 gene in 47 primary cervical carcinomas. HPV status was determined, also by PCR. In 20 of these cases, we examined for loss of heterozygosity (LOH) on chromosome 17p13. In the 47 carcinomas, and in a further 68 biopsy specimens from normal, premalignant and malignant cervix, we investigated aberrant immunocytochemical expression of p53. Immunocytochemically, abnormal p53 expression was detected in 13 of 115 cases (8/57 carcinomas). Somatic mutation in p53 was detected in 1 of 47 cervical carcinomas; 36 were positive for HPV 16, 18 or 33. A low level of allele loss (3 out of 20 cases) was detected on chromosome 17p, occurring in both HPV-positive and HPV-negative cases, and in cases with and without p53 mutations. We conclude that somatic mutation in the hotspot regions of the p53 gene occurs infrequently in cervical carcinomas; that immunocytochemically detectable levels of p53 are also infrequent; and that there is no consistent correlation between p53 mutational status, LOH on chromosome 17p or HPV status in these cancers.     

p15INK4b, p14ARF, and p16INK4a inactivation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors.

PURPOSE: Malignant peripheral nerve sheath tumor (MPNST) can arise sporadically or in association with neurofibromatosis type 1. Deletions at the 9p21 locus have been reported in these tumors. To additionally characterize the status of this chromosomal region, in this study we performed a comprehensive, mostly PCR-based molecular analysis of the three tumor suppressor genes p15(INK4b), p14(ARF) and p16(INK4a) located at the 9p21 locus in 26 cryopreserved MPNSTs. EXPERIMENTAL DESIGN: Fourteen neurofibromatosis type 1-related and 12 sporadic cases were investigated for homozygous deletion coupled with fluorescent in situ hybridization, promoter methylation, and mutational analysis, as well as m-RNA expression. RESULTS: The results showed that an inactivation of one or more genes occurred in 77% of MPNSTs and was mainly achieved through homozygous deletion (46%), which, in turn, encompassed all of the three tandemly linked genes in 83% of the deleted cases. Promoter methylation was at a less extent involved in gene silencing (18%), and no mutations were found. Loss of function at DNA level strongly correlated with loss of mRNA expression accounting for 80% of the cases. Because of the close relationship between p14(ARF) and TP53 and between p15(INK4b)/p16(INK4a) and Rb, these results support a model of a coinactivation of TP53 and Rb pathways in 75% of MPNSTs, with functional consequences on cell growth control and apoptosis. CONCLUSIONS: The inactivation of the 9p21 locus is a frequent and peculiar hallmark of MPNST genetic profile leading also to an impaired apoptosis that could be taken into account in treatment planning of these tumors.     

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.     

p16^INK4a和p14^ARF蛋白在宫颈癌和肺癌中的差异表达

[目的]研究宫颈癌和肺癌中p16INK4a和p14ARF蛋白表达水平的差异及意义。[方法]应用免疫组化方法检测50例宫颈癌和127例肺癌组织中p16INK4a和p14ARF蛋白表达。[结果]50例宫颈癌中,p16INK4a和p14ARF蛋白全部阳性表达;127例肺癌中,p16INK4a和p14ARF蛋白阳性表达率分别为61.42%和30.79%;宫颈癌和肺癌中p16INK4a和p14ARF蛋白表达差异均有显著性（P〈0.01）。[结论]p16INK4a和p14ARF蛋白在宫颈癌中过表达,而在肺癌中缺失表达。提示p16INK4a和p14ARF蛋白在宫颈癌和肺癌的发生发展中所起的作用和作用机制可能不同。     

p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin‐dependent kinases p16^INK4a. In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type‐specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence‐labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT‐PCR were performed, and for p16^INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV‐positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16^INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16^INK4a expression was of statistical significance (p = 0.02). Kaplan‐Meier estimations revealed better survival outcome for patients with HPV‐positive tumors with detectable E6/E7 mRNA and p16^INK4a overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16^INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16^INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA‐positive head and neck carcinomas.     

p16INK4A Expression in Squamous Cell Carcinomas of the Vagina and the Vulva in Tunisian Women

Background: The role of p16INK4A expression in uterine cervix cancer is well established. In the remainingfemale lower genital tract cancers, the importance of p16INK4A up-regulation is less clear. In our study, we analyzedthe role of p16INK4A expression and HPV infection in carcinomas of the vulva and the vagina in Tunisian women.Materials and Methods: We conducted a retrospective study of 30 carcinomas including 15 vulvar squamouscell carcinomas (SCCs) and 15 vaginal SCCs. Immunohistochemistry was used to determine p16INK4A expression.HPV detection and typing was by in situ hybridization. Results: p16INK4A expression was detected in 86.7% ofvaginal SCCs with a strong and diffuse immunostaining in 60% of cases, and also in 73.3% of vulvar SCCswith focal immunoreactivity in 53.3% The association between p16INK4A expression and HPV infection wassignificant in vaginal SCCs (p=0.001) but not vulvar SCCs (p>0.05). Conclusions: p16INK4A expression could beused as a useful marker for HPV positivity in vaginal SCCs similar to that described in uterine cervix cancers.However, our data support the presence of 2 different mechanisms for p16INK4A expression in HPV-related andHPV-unrelated vulvar carcinomas.     

The prognostic significance of p16(INK4a)/p14(ARF) locus deletion and MDM-2 protein expression in adult acute myelogenous leukemia

BACKGROUND: The p16(INK4a) locus encodes two distinct proteins, p16(INK4a) and p14(ARF). Although p16(INK4a) and p15(INK4b) are involved in the phosphorylation of the retinoblastoma (Rb) protein, p14(ARF) interacts with the MDM-2 oncoprotein antagonizing its function as a suppressor of p53. The role of deletions of p16(INK4a)/p14(ARF) and p15(INK4b) and expressions of MDM-2 in myeloid leukemias and its influence on prognosis remain unclear. METHODS: The authors analyzed deletions of p16(INK4)/p14(ARF) and p15(INK4b) in 74 adults with acute myeloid leukemia (AML) by Southern blotting. Western blotting was used to determine Rb protein phosphorylation in patients with deletions of p16(INK4)/p14(ARF) and p15(INK4b). Then, they analyzed the levels of MDM-2 protein expression and correlated it with prognosis in an expanded population of 79 adults with AML by immunoblot analysis and solid-phase radioimmunoassay. RESULTS: Deletions of p16(INK4a)/p14(ARF) and/or p15(INK4b) occurred in 4 of 74 patients (5%) (hemizygous in 3, homozygous in 1 patient). Although the complete remission (CR) rate was similar (79% vs. 50%; P = 0.187), CR duration (10 vs. 46 weeks; P < 0.001), event free survival rate (EFS; 6 vs. 85 weeks; P < 0.004) and overall survival rate (11 vs. 86 weeks; P = 0.001) were significantly shorter in patients with deletions of p16(INK4a)/p14(ARF) and/or p15(INK4b). Thirty-seven (47%) of 79 patients studied for MDM-2 showed increased MDM-2 expression. These patients had a significantly shorter EFS rate (50 vs. 64 weeks; P = 0.023) and a trend for shorter CR duration (24 vs. 53 weeks; P = 0.07). Overall survival rate was not significantly different (50 vs. 84 weeks; P = 0.136). CONCLUSIONS: The authors concluded that 1) deletions of p16(INK4a)/p14(ARF) and/or p15(INK4b) occur with low incidence in patients with AML; 2) patients with deletions of p16(INK4a)/p14(ARF) and/or p15(INK4b) have a significantly shorter CR duration, EFS rate, and overall survival rate than do patients without deletions; (3) overexpression of MDM-2 is common in AML and is associated with shorter CR duration and EFS rate. Mechanisms other than p14(ARF) deletion are responsible for MDM-2 overexpression, and this overexpression may play a role in the biology of the disease.     

p14ARF protein expression is a predictor of both relapse and survival in squamous cell carcinoma of the anterior tongue.

PURPOSE: The INK4A-ARF locus at chromosome 9p21 is frequently altered in head and neck squamous cell carcinoma (SCC) and encodes two distinct tumor suppressors, p16(INK4A) and p14(ARF). This study addressed the role of p14(ARF) as a potential prognostic marker in this disease. EXPERIMENTAL DESIGN: p14(ARF) protein expression was assessed by immunohistochemistry in a cohort of 140 patients with SCC of the anterior tongue. Using univariate and multivariate Cox's proportional hazards models, the outcomes examined were time to disease recurrence or death, with or without clinicopathologic covariates, including nodal status, disease stage, treatment status, Ki-67 staining, and molecular markers with known functional or genetic relationships with p14(ARF) (p16(INK4A), p53, pRb, p21(WAF1/CIP1), E2F-1). RESULTS: On multivariate analysis, p14(ARF) positivity (nucleolar p14(ARF) staining and/or nuclear p14(ARF) staining in >/=30% of tumor cells) was an independent predictor of improved disease-free survival (DFS; P = 0.002) and overall survival (OS; P = 0.002). This was further enhanced when p14(ARF) positivity was cosegregated with positive (>/=1%) p16(INK4A) staining (DFS, P < 0.001; OS, P < 0.001). Patients whose cancers were p14(ARF) negative and p53 positive (>50%) had the poorest outcome (DFS, P < 0.001; OS, P < 0.001) of any patient subgroup analyzed. CONCLUSIONS: These data show that in patients with SCC of the tongue, combined nuclear and nucleolar expression of p14(ARF) protein predicts for improved DFS and OS independent of established prognostic markers.     

Alterations of the p16INK4a/p14ARF pathway in clear cell sarcoma

Clear cell sarcoma (CCS) is a very rare soft tissue sarcoma with a poor prognosis. It has become apparent through immunohistochemical, ultrastructural, and microarray analyses that CCS is a soft tissue melanocytic neoplasm. Alterations in the p16INK4a/p14ARF gene are common in malignant melanoma, which is the prototypical melanocytic neoplasm. In the present study, we performed a clinicopathologic analysis and investigated p16 and cyclin D1 expression by immunohistochemistry in 14 cases. Furthermore, we investigated genetic changes of various tumor suppressor genes and an oncogene, including p16INK4a/p14ARF, p53, beta-catenin, and APC, in 11 cases. The 5-year overall survival rate in all the patients was 33.3%. A high mitotic rate was a significant adverse prognostic factor (P = 0.004). Decreased expression of p16 was observed in 4 (28.6%) of 14 cases. Overexpression of cyclin D1 was observed in 9 cases (64.3%). SSCP analysis followed by DNA direct sequencing revealed point mutations of the p16INK4a gene in 2 of 11 cases (18.2%). In addition, one case with the p14ARF mutation and 2 cases with the p53 mutation were observed. None of the cases harbored mutation of the beta-catenin or APC gene. Homozygous deletion of the p16INK4a/p14ARF gene was detected in one case. Methylation-specific PCR did not reveal hypermethylation of the p16INK4a/p14ARF promoter region in any of the cases. Three cases harbored genetic alterations of the p16INK4a/p14ARF gene (27.3%). All tumors with genetic alterations of the p16INK4a/p14ARF or p53 gene showed a high mitotic rate or tumor necrosis. These alterations were considered to be influential in the poor prognosis of CCS patients.     

Genetic status of cell cycle regulators in squamous cell carcinoma of the oesophagus: the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes are major targets for inactivation

We determined inactivation of the CDKN2A (p16(INK4a) and p14(ARF)) gene in 21 cases of oesophageal squamous cell carcinoma (OSCC). The tumours were also analysed for mutations in exons 5-8 and allelic losses in the p53 gene. In addition, we screened the CDKN2B (p15 INK4b), CDKN2C (p18 INK4c), CDK4 and p53R2 genes for mutations in the tumour tissues. Besides concomitant alterations in the CDKN2A and p53 loci in more than half of the cases, our results showed that in 18 OSCC (86%) the CDKN2A (p16(INK4a) and p14(ARF) ) gene was affected through mutations, homozygous/hemizygous deletions and promoter hypermethylation. Eight out of 10 tumours with mutations or promoter hypermethylation specific to the CDKN2A/p16 INK4a gene showed loss of the wild-type allele. One tumour with a single base deletion in the N-terminus (codon 8) of the CDKN2A/p16(INK4a) gene carried a novel germ-line mutation or a rare polymorphism (Ile51Met) in exon 2 of the CDK4 gene. Promoter hypermethylation in the CDKN2A/p14 ARF gene was detected in 11 tumours. In the p53 gene 15 mutations were detected in 14 tumours. We detected an inverse relationship between CDKN2A/p16 INK4a inactivation and frequency of loss of heterozygosity at the p53 locus (OR 0.09, 95% CI 0.01-0.98; Fisher exact test, P-value approximately 0.03). Screening of nine exons of the p53R2 [Human Genome Organisation (HUGO) official name RRM2B] gene resulted in identification of a novel polymorphism in the 5' untranslated region, which was detected in four cases. Our results suggest that the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes involved in the two cell cycle pathways are major and independent targets of inactivation in OSCC.     

CDKN2 in HPV-positive and HPV-negative cervical-carcinoma cell lines

Human cervical cancers frequently contain retinoblastoma protein (Rb) that is inactivated by binding with human papilloma virus (HPV) E7 protein or through mutation. The CDKN2 gene encodes p 16^INK4 which inhibits cdk4-cyclin D phosphorylation of Rb, preventing the G₁-S transition. To determine whether abnormalities of CDKN2 occur in cervical-cancer cells, 11 cervical cell lines, including 8 HPV-positive cell lines, 2 HPV-negative cell lines containing mutant Rb, and one tumorigenic cell line derived from normal cervical cells following transfection with HPV-16 and v-H-ras (CX16-2HR), were analyzed. No cell line had a homozygous deletion of exon 1 or 2 of CDKN2, and only one cell line, CX16-2HR, had an altered DNA sequence, which represents a common polymorphism at codon 148. To exclude the possibility of other subtle inactivating mutations, immunoblot analysis of protein lysates was performed using a polyclonal anti-p 16^INK4 rabbit anti-serum. Abundant levels of normal-sized pl6^INK4 were observed in all cell samples. Thus, no alterations of CDKN2 were detected in these cervical cell lines. These results confirm that mutational inactivation of p 16^INK4 is a rare event in tumor samples with compromised Rb activity.     

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.