Polymerase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts.
Recently, the inhibitor of cyclin-dependent kinase 4 (CDK4I; also known as p16INK4, multiple tumor suppressor 1, or CDKN2 gene) has been mapped to 9p21 and shown to be mutated or deleted in a large fraction of cell lines derived from many tumor types, including melanoma, suggesting that this gene could be a melanoma suppressor gene.
Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas.
We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families).
These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma.
In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma.
In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma.
These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.
In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma-predisposing gene.
The gene for cyclin-dependent kinase 4, CDK4, has been found in mutated form in the germline from individuals belonging to 2 melanoma kindreds in the United States.
Surprisingly, at the DNA level alone, 96% (43/45) of melanoma cell lines examined were found to be deleted/mutated/methylated for CDKN2A (34/45), homozygously deleted for CDKN2A's neighbor and homolog CDKN2B (6/45), and/or mutated/amplified for CDK4 (5/45).
In addition to the INK4a locus, other genes involved in melanoma development are discussed here, in particular those genes that participate in the same functional pathway, such as CDK4 and Rb, and p53, which is regulated by the alternative product of INK4a.
: The entire CDKN2A coding region and exon 2 of the CDK4 gene of an affected member of each of 52 families from southern Sweden with at least two cases of melanoma in first- or second-degree relatives were screened for mutations by use of polymerase chain reaction-single-strand conformation polymorphism analysis.Statistical tests were two-sided.
Rb phosphorylation is mediated by cyclin-dependent kinases (CDKs), whose activity is enhanced by cyclins and inhibited by CDK inhibitors. p16(INK4A) is a member of a family of inhibitors specific for CDK4 and CDK6. p16(INK4A) is deleted and inactivated in a wide variety of human malignancies, including familial melanomas and pancreatic carcinoma syndromes, indicating that it is an authentic human tumor suppressor.
We hypothesized that germline mutations in the p16INK4A, p14ARF or CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma.