A predisposition gene for familial melanoma has been localized to chromosome bands 9p2l—p22 by linkage analysis in several cohorts of pedigrees (1—4).Positional cloning of a candidate tumor suppressor gene in this region centromeric of the JFNA

The CDKN2Agene maps to chromosome 9p21—22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifi cally to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDK.N2A is homozygously deleted or mutated in a large propor tion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2Aand elucidate the mechanisms of pl6 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A,and an additional 15(25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A.p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipi tation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carded wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indi cated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2Atranscript but not p16. Presumably, the message seen by Northern blotting in these cell lines Is the result of cross-hybridization of the total cDNA probe with the exon lfi transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional pl6, m.icrosatelliteanalysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus sug gesting that this pathway is a primary genetic target in melanoma devel opment.