p16INK4A and p19ARF act in overlapping pathways in cellular immortalization

The INK4A locus encodes two independent but overlapping genes, p16(INK4A) and p19(ARF), and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16(INK4A) or p19(ARF). Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16(INK4A) or p19(ARF) antisense constructs. In immortal derivatives of cell lines expressing antisense p16(INK4A) or p19(ARF) RNA, growth arrest induced by recovery of p16(INK4A) expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19(ARF) is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16(INK4A) and p19(ARF) genes act in partly overlapping pathways.