The allele constitution at codon 72 of the p53 gene (CGC-arginine or CCC-proline) plays a major role in inducing apoptosis in p53 mutant cells. To verify this, we determined GC-status, p53-mutations, and p53-loss of heterozygosity (LOH) in a group of 54 squamous cell carcinomas of the head and neck (SCCHN). A novel approach, using a one-step real-time PCR analysis with fluorescent hybridization probes, was applied to detect the GC status in tumors and corresponding blood samples. p53 mutations in exons 4 to 8 were detected by PCR-SSCP-sequencing analysis. Apoptosis was determined immunohistochemically using antibodies against Fas, FasL, p53, Bcl2, and terminal deoxy-transferase-mediated dUTP nick end labeling (TUNEL) staining. The overall frequency of p53-LOH in SCCHN was 45.2%. In cases of LOH, there was a preferential loss of the proline allele, which was associated with an up-regulation of Bcl2 and lack of co-expression of Fas/FasL and, thus, impaired apoptosis (P < 0.001). Apoptosis was not observed in tumors carrying the arginine allele. p53 mutations were detected in 29.6% of SCCHN and preferentially occurred at the arginine allele (P = 0.01). p53 alterations were more frequently observed in tumors of the oral cavity, oropharynx and hypopharynx, whereas they were rare in larynx carcinomas (P = 0.07). The p53-LOH status was not found to be significantly correlated with sex, age, TNM-status, or tumor grading. We conclude that apoptosis is correlated with the allelic status of codon 72 in SCCHN. Homozygous proline 72 appears to be an important regulator of apoptosis via the Fas/FasL pathway in SCCHN.
Recent studies have shown that most Dutch families with atypical multiple-mole melanoma (FAMMM) have a 19bp germline deletion (p16-Leiden) in exon 2 of the p16 gene (p16 [MIM 600160]) (Gruis et al. 1995; van der Velden et al. 1999van der Velden et al. , 2001. Incomplete penetrance and variable clinical expression in p16-Leiden carriers point to the fact that other genetic mechanisms can compensate for the p16 loss of function (Gruis et al. 1995). Indeed, van der Velden et al. (2001) reported in the Journal that variants in the melanocortin-1 receptor modify the risk of melanoma in p16-Leiden carriers. It is interesting that, apart from reports on simultaneous pancreatic tumors, other cancer types have never been found in such families with p16- Leiden (Gruis et al. 1995).Recently, we found a hereditary heterozygous p16-Leiden mutation in a man who neither smoked more than five cigarettes daily nor abused alcohol, initially diagnosed at age 54 years, who simultaneously developed three carcinomas of the pharynx and oral cavity. The patient showed a familial accumulation of tumor diseases. Both of his parents and his only sister died of cancer very early (the mother of gynecologic cancer, the father of liver carcinoma, and the sister of leukemia). Dutch relatives are not known.DNA from tumors and blood was extracted according to a standard protocol (Sambrook et al. 1989). Mutation analysis of exon 1 and exon 2 of the p16 gene was done by PCR-SSCP sequencing as described by Schneider-Stock et al. (1998).The p16-Leiden mutation was found in the heterozygous constitution in all three tumors and in the blood of the patient. We investigated all DNA samples for p16 promotor methylation to check the status of the retained wild-type allele and, thus, to assess the real functional significance of this finding. The methylation status of the p16 promotor was determined by methylation-specific PCR (Herman et al. 1996). The primer sequences for the unmethylated reactions were (sense) 5 -TTA TTA
as no identifying information about participants was investigated in the study. The study was approved by the Danish Data Protection Agency, the local hospital management and the legal department at Odense University Hospital (permit number 20/15307).
This is the first report on p16-Leiden mutation in head and neck cancer. We provide evidence that the somatic methylation of p16 promotor is associated with the germline transmission of p16-Leiden mutation. This is an example for the rare event of in which aberrant methylation acting as the 'second hit' in a familial cancer syndrome. Our results show that this epigenetic event is equivalent to genetic alterations (mutation/LOH) confirming the Knudson's hypothesis for tumor suppressor gene inactivation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.