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.
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 p21Waf1/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, mu-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN2 did not appear to increase p53 mRNA in WI-38 cells. Purified mu-calpain required only 1 to 3 microM 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.
Zhang
W
W
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo 43699, USA.
Lu
Q
Q
Xie
Z J
ZJ
Mellgren
R L
RL
D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D016196:S Phase / Q000187:drug effects* / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects* / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D016755:Proto-Oncogene Proteins c-jun; D016159:Tumor Suppressor Protein p53; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; D004533:Egtazic Acid; D003978:Diazomethane; D002154:Calpain; D002118:Calcium