1. ¹Genomics Research Center, Academia Sinica, Taipei, Taiwan
2. ²Department of Pediatrics/Hematology-Oncology, University of California, San Diego, CA, USA
3. ³Department of Chemistry, The Ohio State University, Columbus, OH, USA

Correspondence: Dr AL Yu, Genomics Research Center, Academia Sinica, 128, Academia Road, Sec. 2, Nankang Dist., Taipei 115, Taiwan. E-mail: aliceyu@ucsd.edu

⁴These two authors contributed equally to this work.

Received 20 December 2006; Revised 27 March 2007; Accepted 2 April 2007; Published online 7 May 2007.

Abstract

The INK4A locus encodes two tumor suppressor genes, p16^INK4A and p14^ARF, transcribed using alternative exons 1 or 1 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, 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 cDNA revealed that p16 was identical to p16^INK4A, except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16 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 confirmed that p16, like p16^INK4A, is also an ankyrin-repeat protein. Functional analysis of p16 revealed that p16 protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16 repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16^INK4A. Moreover p16, like p16^INK4A, induced cell-cycle arrest at G₀/G₁, and inhibited cell growth in colony formation assay.