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, p16INK4A and p14ARF, transcribed using alternative exons 1α or 1β spliced onto the same exons 2 and 3. Both p16INK4A and p14ARF are capable of inhibiting the cell-cycle progression, albeit in different manner; p16INK4A is phosphorylation of retinoblastoma (pRB) dependent while p14ARF is p53-dependent. In this study, we report the discovery of a novel variant of p16INK4A, termed p16γ, 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 p16γ cDNA revealed that p16γ was identical to p16INK4A, except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16γ expression was detected in the majority of p16INK4A-expressing primary T-ALL and B-ALL patient samples and other p16INK4A-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 confirmed that p16γ, like p16INK4A, is also an ankyrin-repeat protein. Functional analysis of p16γ revealed that p16γ protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16γ repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16INK4A. Moreover p16γ, like p16INK4A, induced cell-cycle arrest at G0/G1, and inhibited cell growth in colony formation assay.