INDUCTION OF THE CYCLINE INHIBITOR p21 EXPRESSION BY CYCLOSPORINE: A POSSIBLE MECHANISM FOR ITS ANTIPROLIFERATIVE EFFECTS.

Abstract

674 The mechanism(s) of cyclosporine's (CsA) efficacy has been traditionally explained on the basis of inhibition of IL-2 and more recently, the induction TGF-β. Both the processes involve the inhibition of cell cycle progression. The cell cycle is controlled by cyclins, regulated by their association with cyclin-dependent kinases (cdks) which promote cell division. cdks are inhibited by selective cdk inhibitors, which prevent the cell from entering the cell-cycle. The most studied and significant cell cyclin inhibitor is p21. In this study, we hypothesized that the efficacy of CsA might be due to the hyperinduction of p21. To examine this hypothesis, we studied the effect of CsA on p21 gene and protein expression, we also examined the effect of CsA on the promoter activity of p21 gene. The expression of p21 gene and protein was studied in human lung adenocarcinoma cell line (A549 cells) cultured with or without CsA. RNA and cell lysates from these cells were prepared, mRNA expression for p21 and β-actin was studied using RT-PCR and p21 protein expression was studied by western blot analysis using anti-p21 monoclonal antibody and p21 control. The ethidium bromide stained bands were scanned and the expression of p21 gene was expressed relative to that of β-actin gene. Fig 1A shows a representative of three experiments which indicated a statistically significant (p=0.05) induction of the p21 gene by CsA (CsA treated 0.37 ±.02 vs 0.12 ± 0.05 untreated).Fig. 1B demonstrate the hyperexpression of p21 protein in the cell lysates of A-549 cells treated with CsA as compared to untreated cells. The stimulation of p21 promoter activity by CsA was studied in A-549 cells transfected with different concentrations (1.5-2.5 μg) of p21-luc plasmid DNA. Fig. 2 demonstrates the stimulation of the promoter activity of p21 by CsA. These results indicate that CsA induces p21 gene and protein and also stimulates its promoter activity. Interestingly, TGF-β has also been shown to induce p21 gene expression and stimulate its promoter activity. This data may suggest the possibility of another mediator for the anti-proliferative of CsA.Fig. 1A Induction of p21 gene by CsAFig. 1B Induction of p21 protein by CsAFig.2 CsA induces the promoter activity of p21 gene