Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells

Eur J Biochem. 1998 Oct 1;257(1):242-8. doi: 10.1046/j.1432-1327.1998.2570242.x.

Abstract

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apoptosis*
  • Base Sequence
  • Carcinoma, Hepatocellular / enzymology
  • Carcinoma, Hepatocellular / metabolism*
  • Carcinoma, Hepatocellular / pathology
  • Caspase 3
  • Caspases / metabolism*
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclin-Dependent Kinases / metabolism
  • Cyclins / metabolism*
  • DNA Primers
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Ginsenosides*
  • Humans
  • Hydrolysis
  • Liver Neoplasms / enzymology
  • Liver Neoplasms / metabolism*
  • Liver Neoplasms / pathology
  • Molecular Sequence Data
  • Saponins / pharmacology
  • Sequence Homology, Amino Acid
  • Staurosporine / pharmacology
  • Tumor Cells, Cultured

Substances

  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • DNA Primers
  • Enzyme Inhibitors
  • Ginsenosides
  • Saponins
  • ginsenoside Rh2
  • Cyclin-Dependent Kinases
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Staurosporine