p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.

1,25-dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08-2.0 micrograms/ml) and of a new vitamin D analogue, the epi-20-analogue KH1060 (0.4-2.0 micrograms/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single-dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse-labelling with BrdUrd. Both calcitriol and KH1060 induced a dose- and time-dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G2 phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti-proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.

Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.