INK4d-Deficient Mice Are Fertile Despite Testicular Atrophy

Abstract

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16^INK4a, is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lackingINK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlikeINK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.

ACKNOWLEDGMENTS

We thank Paula Cohen (Albert Einstein University, New York, N.Y.) for help in the identification of testicular cells, Sandra d'Azzo for suggestions in initiating this project, members of the Roussel and Sherr laboratory for helpful discussions, Esther Latres and Maria Barbacid for communicating unpublished results, and John Cleveland for critical review of the manuscript. We also thank Anne-Marie Hamilton and Suzette Wingo for the characterization of B and T cells and Joseph Watson and Manjula Paruchuri for excellent technical assistance. We are indebted to Catherine Poquette, Xialong Luo, and James Boyette from the Department of Biostatistics at St. Jude Children's Research Hospital for statistical analysis.

This work was supported in part by NIH grant PO1 CA-71907, Cancer Core Grant CA-21765, and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital. C.J.S. is an investigator of the Howard Hughes Medical Institute.