Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse.

In an effort to develop a method for better local control of advanced ovarian cancers, we have established a peritoneal tumor model of ovarian cancer in the nude mouse and applied intraperitoneal gene therapy with the recombinant adenoviral-mediated wild-type p53 tumor suppressor gene (Avp53). The results indicate that: (a) the recombinant adenoviral vector system effectively infected the tumor and normal cells in the peritoneal cavity; and (b) Avp53 treatment effectively suppressed the growth of peritoneal tumors and prolonged the survival of the treated group, especially when the tumor burden was less. These results suggest that intraperitoneal gene therapy using Avp53 is potentially useful as an adjuvant therapeutic modality in human ovarian cancer.