Human p16γ, a novel transcriptional variant of p16INK4A, coexpresses with p16INK4A in cancer cells and inhibits cell-cycle progression

The INK4A locus encodes two tumor suppressor genes, p16(INK4A) and p14(ARF), transcribed using alternative exons 1 alpha or 1 beta spliced onto the same exons 2 and 3. Both p16(INK4A) and p14(ARF) are capable of inhibiting the cell-cycle progression, albeit in different manner; p16(INK4A) is phosphorylation of retinoblastoma (pRB) dependent while p14(ARF) is p53-dependent. In this study, we report the discovery of a novel variant of p16(INK4A), termed p16 gamma, in a primary T-cell acute lymphoblastic leukemia (T-ALL) patient sample and a neuroblastoma cell line, which was expressed at both the transcriptional and translational levels. Cloning and sequencing of the p16 gamma cDNA revealed that p16 gamma was identical to p16(INK4A), except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16 gamma expression was detected in the majority of p16(INK4A)-expressing primary T-ALL and B-ALL patient samples and other p16(INK4A)-expressing tumor samples, but was only barely detectable in some normal mononuclear cells and other non-tumor samples. Structural analysis by nuclear magnetic resonance and circular dichroism con. firmed that p16 gamma, like p16(INK4A), is also an ankyrin-repeat protein. Functional analysis of p16 gamma revealed that p16 gamma protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16 gamma repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16(INK4A). Moreover p16 gamma, like p16(INK4A), induced cell-cycle arrest at G(0)/G(1), and inhibited cell growth in colony formation assay.