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) -alpha and -gamma are down-regulated during skin
tumor progression. The transduction of v-ras(Ha) into primary mouse keratinocytes is sufficient to reduce both RARalpha and RARgamma
protein levels as well as inhibit their transactivation functions. Our primary objective is to investigate the roles that RARalpha and RARgamma play in keratinocyte
tumor cell proliferation. Through retroviral gene transduction, we overexpressed RARalpha or RARgamma into neoplastic mouse epidermal cells with down-regulated endogenous RAR
proteins. Following
all-trans retinoic acid (RA) treatment, RARalpha- and RARgamma-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 RARalpha and RARgamma cause a decrease in S-phase population, while only RARalpha causes a simultaneous G(0)/G(1) block as evidenced by reduced [(3)H]-
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 RARalpha causes a simultaneous sharp, brief increase in the CDKI p16
protein. A later decrease in
cyclin D(1)
protein is also evident in RARalpha- and RARgamma-transduced cells.
Chromatin condensation and PARP cleavage are observed in both RARalpha- and RARgamma-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)-eta. These results suggest that RARalpha and RARgamma 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.