Retinoic acid causes cell growth arrest and an increase in p27 in F9 wild type but not in F9 retinoic acid receptor β 2 knockout cells

Abstract

We have previously shown that an F9 teratocarcinoma retinoic acid receptor β 2 (RARβ 2) knockout cell line exhibits no growth arrest in response to all- trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARα −/−, and F9 RARγ −/− cell lines do growth arrest in response to RA. To examine the role of RARβ 2 in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARβ 2 −/− cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARβ 2 −/− cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARβ 2 −/− cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARβ 2 −/− cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARβ 2 −/− cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARβ 2 is associated with the process of carcinogenesis and RARβ 2 can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARβ 2 mediates these growth inhibitory effects of RA in F9 cells.