PKC regulation of the cyclin dependent kinase (cdk) inhibitor p21 in normal kidney versus kidney cancer cells
Purpose: The cdk inhibitor p21 (cip1/waf1) induces cell cycle arrest through inhibition of cdk/cyclin complexes. p21 expression is induced by p53-dependent and p53-independent pathways. In this study, the role of protein kinase C (PKC) regulation on p21 expression was investigated in normal human kidney (HK-2) versus renal cell carcinoma (RCC) (SN12K-1).
Methods: Human leukaemia U937 cells were used to verify methods. HK-2 and U937 were p53-negative and SN12K1 p53-positive. Treatments of HK-2, SN12K-1 and U937: (1) Pre-treatment with PKC inhibitor H7 (1-5-isoquinolinesulfonyl-2-methylpiperazine) for 30mins, followed by PKC promoter PMA (phorbol-12-myristate-13-acetate) for 24hrs; (2) Pre-treatment with PMA (4hrs), followed by H7 for 1-5hrs. Western blotting and densitometry were used to analyse p21 expression and phospho-PKC (to verify activation of PKC).
Results: (1) HK-2: p21 was expressed in controls. 30mins treatment with H7 had no significant effect on p21, but by 24hrs, p21 had decreased. PMA induced a peak in p21 at 4hrs, but this returned to normal levels by 24hrs. The peak in p21 was not down-regulated significantly by H7. (2) SN12K-1: p21 was expressed in controls. PMA markedly increased p21 (4 and 24hrs). Pre-incubation with H7 before PMA did not inhibit p21 and PMA-induced p21 was not inhibited by H7.
Conclusions: PKC activation may be needed to initiate p21, but not needed to control p21 after its initiation. p21 expression induced by PKC lasts longer in RCC SN12K-1 cells than in normal renal HK-2. This, and the difference in endogenous p53 levels, may help explain resistance of RCCs to treatment.