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 G₁ to S-phase transition

Abstract

The effect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN₂), on the serum-stimulated 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 G₁ cells treated with ZLLY-CHN₂. The content of p21^Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN₂-sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca²⁺ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G₁ phase, μ-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN₂ did not appear to increase p53 mRNA in WI-38 cells. Purified μ-calpain required only 1 to 3 μM Ca²⁺ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN₂ inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G₁.