POINT MUTATIONS CAN INACTIVATE IN-VITRO AND IN-VIVO ACTIVITIES OF P16(INK4A) CDKN2A IN HUMAN GLIOMA/

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malign ant gliomas. Structural and functional evidence has suggested that hom ozygous deletion involving CDKN2A (the genetic locus encoding the cycl in-dependent kinase inhibitor p16(INK4a)) is mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it di fficult to be certain that the CDKN2A gene is their actual target. Her e, we tested this hypothesis by determining the growth suppressive eff ects, cell cycle inhibitions, and the activities of seven naturally oc curring glioma-derived CDKN2A alleles carrying point mutations and fou nd that two of them were functionally compromised. To resolve discrepa ncies among the different existing functional assays, we developed an assay for p16(INK4a) function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating muta tions, prevents pRB phosphorylation in vivo in human glioma cells. The se data suggest that CDKN2A is a critical target for mutational inacti vation in human malignant gliomas.