Functional reassessment of P16 variants using a transfection-based assay

CDKN2A appears to be the major melanoma susceptibility gene, and is also mutated/deleted in sporadic tumours of various types including melanoma. Thus far most approaches to assessing the functionality of mutations in this gene have used in vitro methods such as CDK4 binding and kinase inhibition assays, with sometimes disparate conclusions about functional significance of some variants between studies. We have used a melanoma cell line (MM96L) with no functional p16, as the basis for a "semi-in vivo" transfection-based assay for exogenous p16 functionality based on the growth parameters of the cells and the behaviour of variant proteins after transfection of different CDKN2A cDNAs. Colony counts performed on these transfectants revealed that all but the wild type, + 24 bp ad A148T variants have a diminished ability to inhibit cell growth. All other variants detected either constitutionally in familial melanoma patients (I49T, R87P, G101W and V126D) or somatically in melanomas (N71S, and P81L), appeared functionally impaired in this assay. This diminution of function was independent of CDK4 and CDK6 binding ability. Furthermore, the predominant localization of these variants within the cell was different from that of wt p16. This mislocalization may provide an explanation for their lack of function, or alternatively, it may also be an indicator that the cells are processing unstable, misfolded p16 proteins. This novel assay for assessment of functionality of p16 variants may better reflect the role of some of these mutations in vivo, and as such is a useful adjunct to other in vitro assays.