Overexpression of retinoic acid receptors alpha and gamma into neoplastic epidermal cells causes retinoic acid-induced growth arrest and apoptosis

Abstract

Retinoids are essential for normal epidermal differentiation and are used for the prevention and treatment of numerous skin disorders and cancers in humans. In previous studies, we have shown that retinoic acid receptors (RARs) -α and -γ are down-regulated during skin tumor progression. The transduction of v-rasHa into primary mouse keratinocytes is sufficient to reduce both RARα and RARγ protein levels as well as inhibit their transactivation functions. Our primary objective is to investigate the roles that RARα and RARγ play in keratinocyte tumor cell proliferation. Through retroviral gene transduction, we overexpressed RARα or RARγ into neoplastic mouse epidermal cells with down-regulated endogenous RAR proteins. Following all-trans retinoic acid (RA) treatment, RARα- and RARγ-transduced cell lines exhibit a progressive, dose-dependent growth inhibition relative to the control LXSN cell lines. Further characterization of RAR-transduced cells following RA treatment reveals that both RARα and RARγ cause a decrease in S-phase population, while only RARα causes a simultaneous G0/G1 block as evidenced by reduced [3H]-thymidine incorporation and flow cytometric analysis of DNA content. Following RA treatment, both receptors cause an early, transient increase in the cyclin-dependent kinase inhibitor (CDKI) p21 proteins, while only RARα causes a simultaneous sharp, brief increase in the CDKI p16 protein. A later decrease in cyclin D1 protein is also evident in RARα- and RARγ-transduced cells. Chromatin condensation and PARP cleavage are observed in both RARα- and RARγ-transduced cells indicating an RA-induced apoptosis that may be caspase dependent. Furthermore, both receptors cause a late upregulation and apparent cleavage of the squamous differentiation marker protein kinase C (PKC)-η. These results suggest that RARα and RARγ enhance growth suppression and apoptosis of neoplastic epidermal keratinocytes. This growth inhibitory effect of both retinoid receptors in neoplastic keratinocytes may be achieved through distinct as well as overlapping mechanisms of cell cycle control.