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Tumor Suppressor Gene p16/INK4A Dependent Regulation of Cell Cycle Exit in a Spontaneous Canine Model of Breast Cancer Tumor Suppressor Gene p16/INK4A Dependent Regulation of Cell Cycle Exit in a Spontaneous Canine Model of Breast Cancer

Author

Agarwal, Payal
Sandey, Maninder
DeInnocentes, Patricia
Bird, R. Curtis

Abstract

16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1,leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cellcycle that becomes defective in cancer cells. Well characterized p16-defective canine mammary cancer cell lines (CMT28, CMT27, andCMT12), derived stably p16-transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF),were used to investigate expression of p16 after serum starvation into quiescence followed by re-feeding to induce cell cycle re-entry. Theparental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16-associated proteins,including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cellcycle re-entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells andconfirmed this by3H-thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16-transfected CMT27A andCMT27H cells exited cell cycle post-serum-starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressedupregulated levels of p27 and p16 mRNA, post-serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence anddifferentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 duringquiescence, suggests a potential role for p16 in maintaining these non-proliferative states.

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