The Cyclin-Dependent Kinase Inhibitor and Tumor Suppressor Locus p16/INK4A- p14ARF and Regulation of the Transition Into and Out of the Cell Cycle

Abstract

p16/INK4A/CDKN2A is an important tumor suppressor gene located in the INK4A/ARF locus, which encodes a 16 kDa protein known as p16, and a 14 kDa protein known as p14ARF in humans. p16 arrests cell cycle in early G1 phase thereby inhibiting the binding of cyclin dependent kinase 4/6 with cyclinD1. This leaves the retinoblastoma protein (pRb) tumor suppressor hypo-phosphorylated and S phase transcription factor E2F bound and inactive. p14ARF expression up-regulates cyclin dependent kinase inhibitor p21, which inhibits the G1/S phase transition by stabilizing p53 expression upon disassociation from mdm2. We hypothesized that p16 has a role in exit from the cell cycle, becomes defective in cancer cells and has binding partners other than CDK4/CDK6 in quiescent or differentiated cells when their canonical target proteins are thought to be nonfunctional. We have hypothesized that INK4A/ARF encoded proteins perform important regulatory roles that are defective in canine mammary cancer and may cause loss of differentiation potential. Well characterized p16-defective canine mammary cancer cell lines, normal canine fibroblasts, and CMT-derived p16-transfected CMT cell clones, are used to investigate expression of p16 after serum starvation into quiescence followed by re-feeding to induce cell cycle re-entry. We have successfully demonstrated cell cycle arrest and synchronous cell cycle re-entry in CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells, which is confirmed by 3H-thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. NCF, CMT27A, and CMT28F cells expressed up-regulated levels of p27 mRNA coincidently with elevated expression of p16 mRNA, as cells exited cell cycle and entered quiescence. To find alternate binding partners of p16, co-immunoprecipitation was performed in quiescent CMT27A cells, which resulted in the unique co-immunoprecipitation of the p53-associated and putative tumor suppressor 14-3-3σ protein only in quiescent CMT27A cells in comparison to exponential cells. Levels of 14-3-3σ mRNA expression also rose along with p16 in quiescent NCF cells. We differentiated 3T3-L1 fibroblasts into adipocytes for investigating the p16 and related gene expression profiles during differentiation. This study is the first to report the predicted mRNA and protein sequence of canine p14ARF and p14ARF mRNA expression in canine cells.