The G 1 /S checkpoint of the cell cycle is regulated by p16, p53 and RB tumor suppressor genes. Loss of expression of the p16 INK4 tumor suppressor protein, the product of the CDKN2 gene, has been associated with a wide variety of human malignancies. Mutations, loss of heterozygosity and deletions of the CDKN2 locus have been reported in sporadic and familial cutaneous malignant melanomas (CMM). To investigate the role of the alterations of p16 expression in melanoma, we evaluated by immunohistochemistry the p16 expression and cell proliferation in 79 primary CMM and 10 benign melanocytic nevi (BMN). Forty-six melanomas (58%) and all BMN were found to be p16 positive; 33 melanomas (42%) were considered p16 negative. The extent of invasion according to Clark was significantly higher in p16-negative tumors than in p16-positive tumors. Cell proliferation as expressed by the proportion of positive cells in Ki-67 immunostaining was found to be significantly higher in p16-negative tumors than in p16-positive tumors, although there was no significant difference in the mitotic index between p16-positive and p16-negative tumors. In p16-positive tumors, the number of Ki-67-positive cells correlated with the mitotic index; in p16-negative tumors, there was no correlation between these parameters. Our data suggest that loss of p16 expression is more common in advanced melanomas, and that G 1 /S checkpoint regulation is disrupted in p16-negative melanomas. Our results show that loss of p16 expression is a common event in primary melanomas, which further substantiates the role of p16 as a major tumor suppressor. Int. J. Cancer 74:255-259, 1997.r 1997 Wiley-Liss, Inc.The progression of mammalian cells from the G 1 phase of the cell cycle to the S phase is regulated by a cascade of protein-protein interactions and protein phosphorylations. During G 1 , the D-type cyclins (cyclin D1, D2 and D3) accumulate and form complexes with CDK4 and CDK6. Cyclin D-CDK4/6 complexes phosphorylate the retinoblastoma protein (pRB), which is found in its hypophosphorylated form during G 1 . The phosphorylation of pRB releases a group of transcription factors essential for progression to S phase and onset of DNA replication. p16 INK4 , the protein product of the CDKN2 gene, is thought to be induced by some of these transcription factors to dissemble and inactivate the cyclin D-CDK4/6 complexes. p16 might also be induced earlier in G 1 by various anti-proliferative signals, arresting the cell in G 1 and thus acting as a powerful G 1 /S checkpoint regulator (reviewed by Grana and Reddy, 1995;Sherr and Roberts, 1995).The growth-inhibitory function of p16 makes it a candidate tumor suppressor. Loss of heterozygosity (LOH), homozygous deletion or mutations of the p16 gene have been found in cell lines established from malignant melanoma; mesothelioma; leukemia; glioma; carcinomas of lung, colon, liver, kidneys, esophagus, pancreas, breast, bladder, ovaries and prostate; and various sarcomas (Liu, Q. et al., 1995;Okamoto et al., 1994). Although it has ...