Selective induction of apoptosis in hep 3B cells by topoisomerase I inhibitors: Evidence for a protease-dependent pathway that does not activate cysteine protease P32

Philip N. Adjei, Scott H. Kaufmann, Wai Yee Leung, Fei Mao, Gregory J. Gores

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Abstract

Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappointing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would induce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poison camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In contrast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoisomerase II-DNA complexes. Unexpectedly, CPT- induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, polypeptides that are commonly cleaved in other cell types undergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1β- converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val- Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that (a) apoptosis is induced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisomerase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE-like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type.

Original languageEnglish (US)
Pages (from-to)2588-2596
Number of pages9
JournalJournal of Clinical Investigation
Volume98
Issue number11
DOIs
StatePublished - Dec 1 1996

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Keywords

  • CPP32
  • camptothecin
  • etoposide
  • interleukin-1β-converting enzyme
  • poly(ADP- ribose) polymerase

ASJC Scopus subject areas

  • Medicine(all)

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