Research Article

Disruption of A Regulation Causes Immortalization and Dysregulation of D-Type

Kiran S. Nadella1 and Lawrence S. Kirschner1,2

1Human Cancer Genetics Program and 2Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, The Ohio State University, Columbus, Ohio

Abstract of the thyroid and adrenal glands (5, 6), indicating that it is a tissue- Phosphorylation is a key event in cell cycle control, and specific tumor suppressor . dysregulation of this process is observed in many tumors, Because patient samples are rare, we have generated a mouse including those associated with specific inherited neoplasia model to study this condition. Mice homozygous for a null allele of syndromes. We have shown previously that patients with the Prkar1a exhibit embryonic lethality, whereas heterozygotes, like autosomal dominant tumor predisposition their human counterparts, are tumor prone (7–9). To determine the carry inactivating mutations in the PRKAR1A gene, which mechanism by which loss of Prkar1a promotes tumors, we have encodes the type 1A regulatory subunit of chosen to use primary cultures of mouse embryonic fibroblasts (PKA), the cyclic AMP–dependent protein kinase. This defect (MEFs), which lack secondary alterations commonly found in immortalized cell lines. Using Cre-lox technology, we generated was associated with dysregulation of PKA signaling, and À/À genetic analysis has suggested that complete loss of the gene Prkar1a MEFs in vitro and compared them with otherwise may be required for tumorigenesis. To determine the mecha- genetically identical cells. We report that removal of Prkar1a from nism by which dysregulation of PKA causes tumor formation, cells leads to immortalization in the absence of transformation. In we generated in vitro primary mouse cells lacking the Prkar1a probing the molecular basis for this observation, we find that there protein. We report that this genetic disruption of PKA regu- is strong up-regulation of D1, which is likely linked to the lation causes constitutive PKA activation and immortalization immortalized phenotype. This increase in cyclin D1 protein levels of primary mouse embryonic fibroblasts (MEFs). At the occurs independently of other known pathways, suggesting that molecular level, knockout of Prkar1a leads to up-regulation PKA itself functions as a regulator of cyclin D1 protein levels. of D-type cyclins, and this increase occurs independently of other pathways known to increase cyclin D levels. Despite Materials and Methods the immortalized phenotype, known mediators of cellular Generation of Prkar1aÀ/À mouse embryonic fibroblasts. Mice senescence (e.g., p53 and p19ARF) seem to remain intact in À/À carrying a conditional null allele of Prkar1a (8) were crossed, and primary Prkar1a MEFs. Mechanistically, cyclin D1 mRNA levels are MEFs were prepared from embryonic day 13.5 embryos. Each MEF line was not altered in the knockout cells, but protein half-life is derived from an individual embryo and PCR genotyped as described (8). markedly increased. Using this model, we provide the first Cells were cultured in DMEM with 1% penicillin/streptomycin (Invitrogen, direct genetic evidence that dysregulation of PKA promotes Carlsbad, CA) containing 14% fetal bovine serum (Hyclone, South Logan, important steps in tumorigenesis, and that cyclin D1 is an UT). All the experiments presented here were done in two to three essential target of PKA. (Cancer Res 2005; 65(22): 10307-15) independent MEF lines prepared from different litters. Mice used for preparation of MEFs were maintained in accordance with the highest Introduction standards of animal care in accordance with Institutional Laboratory Animal Care and