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British Journal of Cancer logoLink to British Journal of Cancer
. 1995 Apr;71(4):676–683. doi: 10.1038/bjc.1995.134

Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.

D W Shen 1, S Akiyama 1, P Schoenlein 1, I Pastan 1, M M Gottesman 1
PMCID: PMC2033730  PMID: 7710928

Abstract

Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.

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Selected References

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  1. Andrews P. A., Howell S. B. Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. Cancer Cells. 1990 Feb;2(2):35–43. [PubMed] [Google Scholar]
  2. Behrens B. C., Hamilton T. C., Masuda H., Grotzinger K. R., Whang-Peng J., Louie K. G., Knutsen T., McKoy W. M., Young R. C., Ozols R. F. Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues. Cancer Res. 1987 Jan 15;47(2):414–418. [PubMed] [Google Scholar]
  3. Chu G., Chang E. Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA. Proc Natl Acad Sci U S A. 1990 May;87(9):3324–3327. doi: 10.1073/pnas.87.9.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clugston C. K., McLaughlin K., Kenny M. K., Brown R. Binding of human single-stranded DNA binding protein to DNA damaged by the anticancer drug cis-diamminedichloroplatinum (II). Cancer Res. 1992 Nov 15;52(22):6375–6379. [PubMed] [Google Scholar]
  5. Frei E., 3rd, Cucchi C. A., Rosowsky A., Tantravahi R., Bernal S., Ervin T. J., Ruprecht R. M., Haseltine W. A. Alkylating agent resistance: in vitro studies with human cell lines. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2158–2162. doi: 10.1073/pnas.82.7.2158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Godwin A. K., Meister A., O'Dwyer P. J., Huang C. S., Hamilton T. C., Anderson M. E. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3070–3074. doi: 10.1073/pnas.89.7.3070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gottesman M. M., Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385–427. doi: 10.1146/annurev.bi.62.070193.002125. [DOI] [PubMed] [Google Scholar]
  8. Ishikawa T., Ali-Osman F. Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance. J Biol Chem. 1993 Sep 25;268(27):20116–20125. [PubMed] [Google Scholar]
  9. Jekunen A. P., Hom D. K., Alcaraz J. E., Eastman A., Howell S. B. Cellular pharmacology of dichloro(ethylenediamine)platinum(II) in cisplatin-sensitive and resistant human ovarian carcinoma cells. Cancer Res. 1994 May 15;54(10):2680–2687. [PubMed] [Google Scholar]
  10. Kasahara K., Fujiwara Y., Nishio K., Ohmori T., Sugimoto Y., Komiya K., Matsuda T., Saijo N. Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin. Cancer Res. 1991 Jun 15;51(12):3237–3242. [PubMed] [Google Scholar]
  11. Kawai K., Kamatani N., Georges E., Ling V. Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II). J Biol Chem. 1990 Aug 5;265(22):13137–13142. [PubMed] [Google Scholar]
  12. Kimura E., Enns R. E., Thiebaut F., Howell S. B. Regulation of HSP60 mRNA expression in a human ovarian carcinoma cell line. Cancer Chemother Pharmacol. 1993;32(4):279–285. doi: 10.1007/BF00686173. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Loehrer P. J., Einhorn L. H. Drugs five years later. Cisplatin. Ann Intern Med. 1984 May;100(5):704–713. doi: 10.7326/0003-4819-100-5-704. [DOI] [PubMed] [Google Scholar]
  15. Masuda H., Ozols R. F., Lai G. M., Fojo A., Rothenberg M., Hamilton T. C. Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum (II) in human ovarian cancer cell lines. Cancer Res. 1988 Oct 15;48(20):5713–5716. [PubMed] [Google Scholar]
  16. Moscow J. A., Cowan K. H. Multidrug resistance. J Natl Cancer Inst. 1988 Mar 2;80(1):14–20. doi: 10.1093/jnci/80.1.14. [DOI] [PubMed] [Google Scholar]
  17. Newman E. M., Lu Y., Kashani-Sabet M., Kesavan V., Scanlon K. J. Mechanisms of cross-resistance to methotrexate and 5-fluorouracil in an A2780 human ovarian carcinoma cell subline resistant to cisplatin. Biochem Pharmacol. 1988 Feb 1;37(3):443–447. doi: 10.1016/0006-2952(88)90212-2. [DOI] [PubMed] [Google Scholar]
  18. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  19. Pil P. M., Lippard S. J. Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science. 1992 Apr 10;256(5054):234–237. doi: 10.1126/science.1566071. [DOI] [PubMed] [Google Scholar]
  20. Plooy A. C., van Dijk M., Berends F., Lohman P. H. Formation and repair of DNA interstrand cross-links in relation to cytotoxicity and unscheduled DNA synthesis induced in control and mutant human cells treated with cis-diamminedichloroplatinum(II). Cancer Res. 1985 Sep;45(9):4178–4184. [PubMed] [Google Scholar]
  21. Puchalski R. B., Fahl W. E. Expression of recombinant glutathione S-transferase pi, Ya, or Yb1 confers resistance to alkylating agents. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2443–2447. doi: 10.1073/pnas.87.7.2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richon V. M., Schulte N., Eastman A. Multiple mechanisms of resistance to cis-diamminedichloroplatinum(II) in murine leukemia L1210 cells. Cancer Res. 1987 Apr 15;47(8):2056–2061. [PubMed] [Google Scholar]
  23. Scanlon K. J., Kashani-Sabet M. Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells. Proc Natl Acad Sci U S A. 1988 Feb;85(3):650–653. doi: 10.1073/pnas.85.3.650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schoenlein P. V., Shen D. W., Barrett J. T., Pastan I., Gottesman M. M. Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells. Mol Biol Cell. 1992 May;3(5):507–520. doi: 10.1091/mbc.3.5.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shellard S. A., Hosking L. K., Hill B. T. Anomalous relationship between cisplatin sensitivity and the formation and removal of platinum-DNA adducts in two human ovarian carcinoma cell lines in vitro. Cancer Res. 1991 Sep 1;51(17):4557–4564. [PubMed] [Google Scholar]
  26. Shen D. W., Fojo A., Chin J. E., Roninson I. B., Richert N., Pastan I., Gottesman M. M. Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification. Science. 1986 May 2;232(4750):643–645. doi: 10.1126/science.3457471. [DOI] [PubMed] [Google Scholar]
  27. Shen D. W., Lu Y. G., Chin K. V., Pastan I., Gottesman M. M. Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to MRD1 gene expression. J Cell Sci. 1991 Mar;98(Pt 3):317–322. doi: 10.1242/jcs.98.3.317. [DOI] [PubMed] [Google Scholar]
  28. Teicher B. A., Cucchi C. A., Lee J. B., Flatow J. L., Rosowsky A., Frei E., 3rd Alkylating agents: in vitro studies of cross-resistance patterns in human cell lines. Cancer Res. 1986 Sep;46(9):4379–4383. [PubMed] [Google Scholar]
  29. Timmer-Bosscha H., Mulder N. H., de Vries E. G. Modulation of cis-diamminedichloroplatinum(II) resistance: a review. Br J Cancer. 1992 Aug;66(2):227–238. doi: 10.1038/bjc.1992.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ueda K., Clark D. P., Chen C. J., Roninson I. B., Gottesman M. M., Pastan I. The human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation. J Biol Chem. 1987 Jan 15;262(2):505–508. [PubMed] [Google Scholar]
  31. Zhen W., Link C. J., Jr, O'Connor P. M., Reed E., Parker R., Howell S. B., Bohr V. A. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Mol Cell Biol. 1992 Sep;12(9):3689–3698. doi: 10.1128/mcb.12.9.3689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de Jong S., Timmer-Bosscha H., de Vries E. G., Mulder N. H. Effect of novobiocin on cisplatin cytotoxicity and DNA interstrand cross-link formation in a cisplatin-resistant, small-cell lung carcinoma cell line. Int J Cancer. 1993 Jan 2;53(1):110–117. doi: 10.1002/ijc.2910530121. [DOI] [PubMed] [Google Scholar]

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