Mouse p10, an Alternative Spliced Form of p15 INK4b , Inhibits Cell Cycle Progression and Malignant Transformation Ignacio Pe ´rez de Castro, 1,2 Marta Benet, 1 Marı ´a Jime ´nez, 3 Saba Alzabin, 1 Marcos Malumbres, 2 and Angel Pellicer 1 1 Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, New York; 2 Molecular Oncology and 3 Experimental Therapeutics Programs, Centro Nacional de Investigaciones Oncologicas CNIO, Madrid, Spain Abstract The INK4 family of proteins negatively regulates cell cycle progression at the G 1 -S transition by inhibiting cyclin- dependent kinases. Two of these cell cycle inhibitors, p16 INK4A and p15 INK4B , have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16 INK4A , the INK4A locus encodes a splice variant, termed p12— specifically expressed in human pancreas—and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15 INK4B tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15 INK4B , p10 expression is also induced by oncogenic insults and transforming growth factor- h treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression. (Cancer Res 2005; 65(8): 3249-56) Introduction Regulation of cell division is a highly complex process that involves many different pathways. During the last decade, many molecules implicated in cell cycle regulation have been identified (1). Cell cycle regulators include cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors, which are involved in the regulation of cell cycle progression in response to mitogenic and antimitogenic signaling. Progression throughout the cell cycle depends on the sequential activation of CDKs by their cyclin partners. Thus, complexes formed by CDK4 or CDK6 and D-type cyclins phosphorylate the retinoblastoma protein, thereby helping to cancel its growth-repressive functions at the G 1 -S cell cycle checkpoint. CDK4 and CDK6 activities are specifically inhibited by the INK4 family of CDK inhibitors. INK4 family members include p16 INK4A , p15 INK4B , p18 INK4C , and p19 INK4D , and are characterized by the presence of multiple ankyrin repeats, which participate in CDK binding. The importance of the cyclin D/CDK4/INK4/retinoblastoma pathway in cancer is undeniable, since tumor-associated alter- ations in at least one of the proteins of this pathway have been found in >80% of all human malignancies (2). Alterations include translocation, amplification, or overexpression of D-type cyclins or CDKs, mutations in CDK4 and CDK6 (resulting in loss of INK4 binding), as well as inactivation of retinoblastoma or the CDK inhibitors (mainly p16 INK4A and p15 INK4B ). The importance of the INK4A locus in cancer was further underscored after the discovery of an additional protein, p19 ARF (p14 ARF in humans), as a tumor suppressor encoded by an alternative reading frame (3). Whereas cell cycle inhibition by p16 INK4A is mediated by retinoblastoma activation, it was soon realized that ARF exerts its tumor suppression functions through the p53 pathway since ARF stabilizes p53 by negatively regulating mdm2 (4–6). In fact, lack of expression of ARF has been associated with several tumor types (7, 8) and its deletion produces a tumor-prone phenotype in gene- targeted mice (9). Another splice variant of INK4A, termed p12, is specifically expressed in human pancreas (10). This protein does not interact with CDK4 but is capable of suppressing growth in a retinoblastoma protein–independent manner. Similarly, an alternatively spliced form, termed as p10, has been described in the human INK4B locus (11). Like p12, human p10 is synthesized through the use of an alternative splice donor site within intron 1 and retains growth-inhibitory activity despite undetectable CDK4 and CDK6 binding. However, unlike p12, which is specifically expressed in pancreas, human p10 is present in several different normal and tumor human cell lines (11). Here, we describe a functional characterization of the mouse orthologue of human p10. Like its human counterpart, mouse p10 arises from the use of an alternative splice donor site within intron 1 of the INK4B locus. Mouse p10 is widely expressed in murine tissues although to a lesser extent than p15. Like p15 INK4B , p10 expression is also induced by oncogenic insults and transforming growth factor-h (TGF-h) treatment. Interestingly, we show that mouse p10 is able to induce cell cycle arrest and to protect against malignant transformation in vitro in a p53- dependent manner, suggesting a complex biological function for both the INK4A and INK4B loci in regulating the retinoblastoma and p53 pathways. Materials and Methods Plasmids, primers, and probes. A genomic fragment containing the mouse INK4B locus (12) was used to study and characterize the mouse p10 sequence. This 6 kb fragment includes about 700 bp upstream of the Requests for reprints: Ignacio Pe ´rez de Castro, Molecular Oncology Program, Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, Madrid 28029, Spain. Phone: 34-91-224-6900; Fax: 34-91-732-8033; E-mail: iperez@cnio.es. I2005 American Association for Cancer Research. www.aacrjournals.org 3249 Cancer Res 2005; 65: (8). 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