Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Dec 1;98(11):2588–2596. doi: 10.1172/JCI119078

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.

P N Adjei 1, S H Kaufmann 1, W Y Leung 1, F Mao 1, G J Gores 1
PMCID: PMC507717  PMID: 8958222

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-1beta-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.

Full Text

The Full Text of this article is available as a PDF (296.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. A comparison of lipiodol chemoembolization and conservative treatment for unresectable hepatocellular carcinoma. Groupe d'Etude et de Traitement du Carcinome Hépatocellulaire. N Engl J Med. 1995 May 11;332(19):1256–1261. doi: 10.1056/NEJM199505113321903. [DOI] [PubMed] [Google Scholar]
  2. Beidler D. R., Cheng Y. C. Camptothecin induction of a time- and concentration-dependent decrease of topoisomerase I and its implication in camptothecin activity. Mol Pharmacol. 1995 May;47(5):907–914. [PubMed] [Google Scholar]
  3. Boege F., Andersen A., Jensen S., Zeidler R., Kreipe H. Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies. Am J Pathol. 1995 Jun;146(6):1302–1308. [PMC free article] [PubMed] [Google Scholar]
  4. Bruix J., Castells A., Montanyà X., Calvet X., Brú C., Ayuso C., Jover L., García L., Vilana R., Boix L. Phase II study of transarterial embolization in European patients with hepatocellular carcinoma: need for controlled trials. Hepatology. 1994 Sep;20(3):643–650. [PubMed] [Google Scholar]
  5. Bruno S., Del Bino G., Lassota P., Giaretti W., Darzynkiewicz Z. Inhibitors of proteases prevent endonucleolysis accompanying apoptotic death of HL-60 leukemic cells and normal thymocytes. Leukemia. 1992 Nov;6(11):1113–1120. [PubMed] [Google Scholar]
  6. Bruno S., Lassota P., Giaretti W., Darzynkiewicz Z. Apoptosis of rat thymocytes triggered by prednisolone, camptothecin, or teniposide is selective to G0 cells and is prevented by inhibitors of proteases. Oncol Res. 1992;4(1):29–35. [PubMed] [Google Scholar]
  7. Carson D. A., Ribeiro J. M. Apoptosis and disease. Lancet. 1993 May 15;341(8855):1251–1254. doi: 10.1016/0140-6736(93)91154-e. [DOI] [PubMed] [Google Scholar]
  8. Cerretti D. P., Kozlosky C. J., Mosley B., Nelson N., Van Ness K., Greenstreet T. A., March C. J., Kronheim S. R., Druck T., Cannizzaro L. A. Molecular cloning of the interleukin-1 beta converting enzyme. Science. 1992 Apr 3;256(5053):97–100. doi: 10.1126/science.1373520. [DOI] [PubMed] [Google Scholar]
  9. Chang J. Y., Dethlefsen L. A., Barley L. R., Zhou B. S., Cheng Y. C. Characterization of camptothecin-resistant Chinese hamster lung cells. Biochem Pharmacol. 1992 Jun 9;43(11):2443–2452. doi: 10.1016/0006-2952(92)90325-d. [DOI] [PubMed] [Google Scholar]
  10. Chinnaiyan A. M., Orth K., O'Rourke K., Duan H., Poirier G. G., Dixit V. M. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. J Biol Chem. 1996 Mar 1;271(9):4573–4576. doi: 10.1074/jbc.271.9.4573. [DOI] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Colombo M. Hepatocellular carcinoma in cirrhotics. Semin Liver Dis. 1993 Nov;13(4):374–383. doi: 10.1055/s-2007-1007366. [DOI] [PubMed] [Google Scholar]
  13. Dive C., Evans C. A., Whetton A. D. Induction of apoptosis--new targets for cancer chemotherapy. Semin Cancer Biol. 1992 Dec;3(6):417–427. [PubMed] [Google Scholar]
  14. Fernandes-Alnemri T., Litwack G., Alnemri E. S. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J Biol Chem. 1994 Dec 9;269(49):30761–30764. [PubMed] [Google Scholar]
  15. Fernandes-Alnemri T., Litwack G., Alnemri E. S. Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family. Cancer Res. 1995 Jul 1;55(13):2737–2742. [PubMed] [Google Scholar]
  16. Fernandes-Alnemri T., Takahashi A., Armstrong R., Krebs J., Fritz L., Tomaselli K. J., Wang L., Yu Z., Croce C. M., Salveson G. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 1995 Dec 15;55(24):6045–6052. [PubMed] [Google Scholar]
  17. Fisher D. E. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994 Aug 26;78(4):539–542. doi: 10.1016/0092-8674(94)90518-5. [DOI] [PubMed] [Google Scholar]
  18. Fraser A., Evan G. A license to kill. Cell. 1996 Jun 14;85(6):781–784. doi: 10.1016/s0092-8674(00)81005-3. [DOI] [PubMed] [Google Scholar]
  19. Froelich-Ammon S. J., Osheroff N. Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J Biol Chem. 1995 Sep 15;270(37):21429–21432. doi: 10.1074/jbc.270.37.21429. [DOI] [PubMed] [Google Scholar]
  20. Gores G. J., Nieminen A. L., Wray B. E., Herman B., Lemasters J. J. Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death. J Clin Invest. 1989 Feb;83(2):386–396. doi: 10.1172/JCI113896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaufmann S. H. Additional members of the rat liver lamin polypeptide family. Structural and immunological characterization. J Biol Chem. 1989 Aug 15;264(23):13946–13955. [PubMed] [Google Scholar]
  22. Kaufmann S. H., Desnoyers S., Ottaviano Y., Davidson N. E., Poirier G. G. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993 Sep 1;53(17):3976–3985. [PubMed] [Google Scholar]
  23. Kaufmann S. H. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res. 1989 Nov 1;49(21):5870–5878. [PubMed] [Google Scholar]
  24. Kaufmann S. H., McLaughlin S. J., Kastan M. B., Liu L. F., Karp J. E., Burke P. J. Topoisomerase II levels during granulocytic maturation in vitro and in vivo. Cancer Res. 1991 Jul 1;51(13):3534–3543. [PubMed] [Google Scholar]
  25. Kerr J. F., Winterford C. M., Harmon B. V. Apoptosis. Its significance in cancer and cancer therapy. Cancer. 1994 Apr 15;73(8):2013–2026. doi: 10.1002/1097-0142(19940415)73:8<2013::aid-cncr2820730802>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  26. Koch C. J., Giandomenico A. R. The alkaline elution technique for measuring DNA single strand breaks: increased reliability and sensitivity. Anal Biochem. 1994 Jul;220(1):58–65. doi: 10.1006/abio.1994.1299. [DOI] [PubMed] [Google Scholar]
  27. Kohn K. W. DNA filter elution: a window on DNA damage in mammalian cells. Bioessays. 1996 Jun;18(6):505–513. doi: 10.1002/bies.950180613. [DOI] [PubMed] [Google Scholar]
  28. Kostura M. J., Tocci M. J., Limjuco G., Chin J., Cameron P., Hillman A. G., Chartrain N. A., Schmidt J. A. Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5227–5231. doi: 10.1073/pnas.86.14.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kwo P., Patel T., Bronk S. F., Gores G. J. Nuclear serine protease activity contributes to bile acid-induced apoptosis in hepatocytes. Am J Physiol. 1995 Apr;268(4 Pt 1):G613–G621. doi: 10.1152/ajpgi.1995.268.4.G613. [DOI] [PubMed] [Google Scholar]
  30. Lamarre D., Talbot B., de Murcia G., Laplante C., Leduc Y., Mazen A., Poirier G. G. Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study. Biochim Biophys Acta. 1988 Jul 13;950(2):147–160. doi: 10.1016/0167-4781(88)90007-3. [DOI] [PubMed] [Google Scholar]
  31. Li C. J., Wang C., Pardee A. B. Induction of apoptosis by beta-lapachone in human prostate cancer cells. Cancer Res. 1995 Sep 1;55(17):3712–3715. [PubMed] [Google Scholar]
  32. Liu L. F. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem. 1989;58:351–375. doi: 10.1146/annurev.bi.58.070189.002031. [DOI] [PubMed] [Google Scholar]
  33. Lowe S. W. Cancer therapy and p53. Curr Opin Oncol. 1995 Nov;7(6):547–553. doi: 10.1097/00001622-199511000-00013. [DOI] [PubMed] [Google Scholar]
  34. Martin S. J., Green D. R. Protease activation during apoptosis: death by a thousand cuts? Cell. 1995 Aug 11;82(3):349–352. doi: 10.1016/0092-8674(95)90422-0. [DOI] [PubMed] [Google Scholar]
  35. Mattern M. R., Mong S. M., Bartus H. F., Mirabelli C. K., Crooke S. T., Johnson R. K. Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells. Cancer Res. 1987 Apr 1;47(7):1793–1798. [PubMed] [Google Scholar]
  36. Miller D. K., Ayala J. M., Egger L. A., Raju S. M., Yamin T. T., Ding G. J., Gaffney E. P., Howard A. D., Palyha O. C., Rolando A. M. Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells. J Biol Chem. 1993 Aug 25;268(24):18062–18069. [PubMed] [Google Scholar]
  37. Munday N. A., Vaillancourt J. P., Ali A., Casano F. J., Miller D. K., Molineaux S. M., Yamin T. T., Yu V. L., Nicholson D. W. Molecular cloning and pro-apoptotic activity of ICErelII and ICErelIII, members of the ICE/CED-3 family of cysteine proteases. J Biol Chem. 1995 Jun 30;270(26):15870–15876. doi: 10.1074/jbc.270.26.15870. [DOI] [PubMed] [Google Scholar]
  38. Murakami Y., Hayashi K., Hirohashi S., Sekiya T. Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res. 1991 Oct 15;51(20):5520–5525. [PubMed] [Google Scholar]
  39. Nicholson D. W. ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis. Nat Biotechnol. 1996 Mar;14(3):297–301. doi: 10.1038/nbt0396-297. [DOI] [PubMed] [Google Scholar]
  40. Oberhammer F. A., Pavelka M., Sharma S., Tiefenbacher R., Purchio A. F., Bursch W., Schulte-Hermann R. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5408–5412. doi: 10.1073/pnas.89.12.5408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ozturk M. p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet. 1991 Nov 30;338(8779):1356–1359. doi: 10.1016/0140-6736(91)92236-u. [DOI] [PubMed] [Google Scholar]
  42. Patel T., Bronk S. F., Gores G. J. Increases of intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. J Clin Invest. 1994 Dec;94(6):2183–2192. doi: 10.1172/JCI117579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Patel T., Gores G. J., Kaufmann S. H. The role of proteases during apoptosis. FASEB J. 1996 Apr;10(5):587–597. doi: 10.1096/fasebj.10.5.8621058. [DOI] [PubMed] [Google Scholar]
  44. Pelletier G., Roche A., Ink O., Anciaux M. L., Derhy S., Rougier P., Lenoir C., Attali P., Etienne J. P. A randomized trial of hepatic arterial chemoembolization in patients with unresectable hepatocellular carcinoma. J Hepatol. 1990 Sep;11(2):181–184. doi: 10.1016/0168-8278(90)90110-d. [DOI] [PubMed] [Google Scholar]
  45. Planchon S. M., Wuerzberger S., Frydman B., Witiak D. T., Hutson P., Church D. R., Wilding G., Boothman D. A. Beta-lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent response. Cancer Res. 1995 Sep 1;55(17):3706–3711. [PMC free article] [PubMed] [Google Scholar]
  46. Pommier Y., Leteurtre F., Fesen M. R., Fujimori A., Bertrand R., Solary E., Kohlhagen G., Kohn K. W. Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors. Cancer Invest. 1994;12(5):530–542. doi: 10.3109/07357909409021413. [DOI] [PubMed] [Google Scholar]
  47. Puisieux A., Lim S., Groopman J., Ozturk M. Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 1991 Nov 15;51(22):6185–6189. [PubMed] [Google Scholar]
  48. Ringe B., Pichlmayr R., Wittekind C., Tusch G. Surgical treatment of hepatocellular carcinoma: experience with liver resection and transplantation in 198 patients. World J Surg. 1991 Mar-Apr;15(2):270–285. doi: 10.1007/BF01659064. [DOI] [PubMed] [Google Scholar]
  49. Rosser B. G., Powers S. P., Gores G. J. Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach. J Biol Chem. 1993 Nov 5;268(31):23593–23600. [PubMed] [Google Scholar]
  50. Rowinsky E. K., Noe D. A., Grochow L. B., Sartorious S. E., Bowling M. K., Chen T. L., Lubejko B. G., Kaufmann S. H., Donehower R. C. Phase I and pharmacologic studies of pyrazoloacridine, a novel DNA intercalating agent, on single-dosing and multiple-dosing schedules. J Clin Oncol. 1995 Aug;13(8):1975–1984. doi: 10.1200/JCO.1995.13.8.1975. [DOI] [PubMed] [Google Scholar]
  51. Schulte-Hermann R., Bursch W., Kraupp-Grasl B., Oberhammer F., Wagner A. Programmed cell death and its protective role with particular reference to apoptosis. Toxicol Lett. 1992 Dec;64-65 Spec No:569–574. doi: 10.1016/0378-4274(92)90233-a. [DOI] [PubMed] [Google Scholar]
  52. Slagle B. L., Zhou Y. Z., Butel J. S. Hepatitis B virus integration event in human chromosome 17p near the p53 gene identifies the region of the chromosome commonly deleted in virus-positive hepatocellular carcinomas. Cancer Res. 1991 Jan 1;51(1):49–54. [PubMed] [Google Scholar]
  53. Slichenmyer W. J., Nelson W. G., Slebos R. J., Kastan M. B. Loss of a p53-associated G1 checkpoint does not decrease cell survival following DNA damage. Cancer Res. 1993 Sep 15;53(18):4164–4168. [PubMed] [Google Scholar]
  54. Solary E., Bertrand R., Pommier Y. Apoptosis induced by DNA topoisomerase I and II inhibitors in human leukemic HL-60 cells. Leuk Lymphoma. 1994 Sep;15(1-2):21–32. doi: 10.3109/10428199409051674. [DOI] [PubMed] [Google Scholar]
  55. Takahashi A., Alnemri E. S., Lazebnik Y. A., Fernandes-Alnemri T., Litwack G., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8395–8400. doi: 10.1073/pnas.93.16.8395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tanaka S., Toh Y., Adachi E., Matsumata T., Mori R., Sugimachi K. Tumor progression in hepatocellular carcinoma may be mediated by p53 mutation. Cancer Res. 1993 Jun 15;53(12):2884–2887. [PubMed] [Google Scholar]
  57. Tang C., Willingham M. C., Reed J. C., Miyashita T., Ray S., Ponnathpur V., Huang Y., Mahoney M. E., Bullock G., Bhalla K. High levels of p26BCL-2 oncoprotein retard taxol-induced apoptosis in human pre-B leukemia cells. Leukemia. 1994 Nov;8(11):1960–1969. [PubMed] [Google Scholar]
  58. Tewari M., Quan L. T., O'Rourke K., Desnoyers S., Zeng Z., Beidler D. R., Poirier G. G., Salvesen G. S., Dixit V. M. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995 Jun 2;81(5):801–809. doi: 10.1016/0092-8674(95)90541-3. [DOI] [PubMed] [Google Scholar]
  59. Thompson C. B. Apoptosis in the pathogenesis and treatment of disease. Science. 1995 Mar 10;267(5203):1456–1462. doi: 10.1126/science.7878464. [DOI] [PubMed] [Google Scholar]
  60. Uckun F. M., Stewart C. F., Reaman G., Chelstrom L. M., Jin J., Chandan-Langlie M., Waddick K. G., White J., Evans W. E. In vitro and in vivo activity of topotecan against human B-lineage acute lymphoblastic leukemia cells. Blood. 1995 May 15;85(10):2817–2828. [PubMed] [Google Scholar]
  61. Wang L., Miura M., Bergeron L., Zhu H., Yuan J. Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell. 1994 Sep 9;78(5):739–750. doi: 10.1016/s0092-8674(94)90422-7. [DOI] [PubMed] [Google Scholar]
  62. Wingo P. A., Tong T., Bolden S. Cancer statistics, 1995. CA Cancer J Clin. 1995 Jan-Feb;45(1):8–30. doi: 10.3322/canjclin.45.1.8. [DOI] [PubMed] [Google Scholar]
  63. Wozniak A. J., Ross W. E. DNA damage as a basis for 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside) (etoposide) cytotoxicity. Cancer Res. 1983 Jan;43(1):120–124. [PubMed] [Google Scholar]
  64. Yuan J., Shaham S., Ledoux S., Ellis H. M., Horvitz H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell. 1993 Nov 19;75(4):641–652. doi: 10.1016/0092-8674(93)90485-9. [DOI] [PubMed] [Google Scholar]
  65. Zou Y., Horikoshi I., Ueno M., Gu X., Perez-Soler R. Targeting liver tumors by administering liposomal doxorubicin into the hepatic artery. Int J Cancer. 1992 May 8;51(2):232–237. doi: 10.1002/ijc.2910510211. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES