FUNCTIONAL-ANALYSIS OF WILD-TYPE AND MALIGNANT GLIOMA DERIVED CDKN2A-BETA ALLELES - EVIDENCE FOR AN RB-INDEPENDENT GROWTH SUPPRESSIVE PATHWAY

The tumor suppressor gene CDKN2A (p16/MTS1/ 1NK4A), which encodes the cyclin-dependent kinase inhibitor p16(INK4a), is a target of 9p21 dele tions during the malignant progression of human gliomas, This gene als o encodes a second protein product (human p16 beta, murine p19(ARF)), which originates from an unrelated exon of CDKN2A (exon 1 beta) splice d onto exon 2 in an alternate reading frame, Cell cycle arrest by p16 beta is caused by an as yet unidentified pathway, In order to test the candidacy of p16 beta as a glioma suppressor, we replaced p16(INK4a), p15(INK4b) and p16 beta wild-type as well as a series of seven glioma -derived p16 beta alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A(-)/RB+ (U-87MG and U-251MG) or CDKN2A(+)/RB- (LN-319) endogenous backgrounds and demo nstrated that p16 beta can act as a functional glioma cell growth supp ressor, Moreover, p16 beta, but not p16(INK4a) or p15(INK4b) inhibited the growth of RE-negative LN-319 cells, indicating that p16 beta like ly exerts its effects through an RB-independent pathway, In vitro and in vivo assays of pRB phosphorylation were consistent with this interp retation, Since none of the glioma-derived p16 beta mutations inactiva ted their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16(INK4a) a nd p16 beta) likely exclusively target p16(INK4a).