INHIBITION OF THE GROWTH OF WI-38 FIBROBLASTS BY BENZYLOXYCARBONYL-LEU-LEU-TYR DIAZOMETHYL KETONE - EVIDENCE THAT CLEAVAGE OF P53 BY A CALPAIN-LIKE PROTEASE IS NECESSARY FOR G(1) TO S-PHASE TRANSITION

The effect of a calpain-selective cell permeant inhibitor, benzyloxyca rbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN2), on the serum-stimula ted growth of WI-38 human fibroblasts has been investigated, Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase, Protein blotting experiments indicated that p53 immunoreactivity increased in late G(1) cells treated with ZLLY-C HN2. The content of p21(Waf1/Cip1) CDK inhibitor also increased, provi ding a mechanism for the observed failure to enter S-phase, Further st udies indicated that p53 could be degraded by a ZLLY-CHN2-sensitive pr otease immediately prior to S-phase, but that proteolysis did not occu r after this critical time point, Chelation of extracellular Ca2+ by a ddition of EGTA inhibited the p53 degradation, Consistent with proteol ysis of p53 in late G(1) phase, mu-calpain immunoreactivity transientl y accumulated in ceh nuclei at this time, ZLLY-CHN, did not appear to increase p53 mRNA in WI-38 cells, Purified mu-calpain required only 1 to 3 mu M Ca2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN, inhibits progression of WI-38 cells into S-pha se by inactivating a calpain-like protease that is responsible for pro teolysis of constitutively expressed p53 in late G(1).