Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Aug;8(8):3316–3321. doi: 10.1128/mcb.8.8.3316

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

S E Kane 1, B R Troen 1, S Gal 1, K Ueda 1, I Pastan 1, M M Gottesman 1
PMCID: PMC363566  PMID: 2463474

Abstract

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Full text

PDF

Images in this article

3318Image
on p.3318
3318Image
on p.3318
3319Image
on p.3319

Selected References

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

  1. Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Cartier M., Chang M. W., Stanners C. P. Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells. Mol Cell Biol. 1987 May;7(5):1623–1628. doi: 10.1128/mcb.7.5.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chiang T. R., McConlogue L. Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells. Mol Cell Biol. 1988 Feb;8(2):764–769. doi: 10.1128/mcb.8.2.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Fojo A. T., Whang-Peng J., Gottesman M. M., Pastan I. Amplification of DNA sequences in human multidrug-resistant KB carcinoma cells. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7661–7665. doi: 10.1073/pnas.82.22.7661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gal S., Gottesman M. M. Isolation and sequence of a cDNA for human pro-(cathepsin L). Biochem J. 1988 Jul 1;253(1):303–306. doi: 10.1042/bj2530303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gal S., Gottesman M. M. The major excreted protein (MEP) of transformed mouse cells and cathepsin L have similar protease specificity. Biochem Biophys Res Commun. 1986 Aug 29;139(1):156–162. doi: 10.1016/s0006-291x(86)80093-6. [DOI] [PubMed] [Google Scholar]
  9. Gal S., Gottesman M. M. The major excreted protein of transformed fibroblasts is an activable acid-protease. J Biol Chem. 1986 Feb 5;261(4):1760–1765. [PubMed] [Google Scholar]
  10. Gal S., Willingham M. C., Gottesman M. M. Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated. J Cell Biol. 1985 Feb;100(2):535–544. doi: 10.1083/jcb.100.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gottesman M. M., Cabral F. Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts. Biochemistry. 1981 Mar 17;20(6):1659–1665. doi: 10.1021/bi00509a039. [DOI] [PubMed] [Google Scholar]
  12. Gottesman M. M., Sobel M. E. Tumor promoters and Kirsten sarcoma virus increase synthesis of a secreted glycoprotein by regulating levels of translatable mRNA. Cell. 1980 Feb;19(2):449–455. doi: 10.1016/0092-8674(80)90519-x. [DOI] [PubMed] [Google Scholar]
  13. Gottesman M. M. Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2767–2771. doi: 10.1073/pnas.75.6.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kartner N., Riordan J. R., Ling V. Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science. 1983 Sep 23;221(4617):1285–1288. doi: 10.1126/science.6137059. [DOI] [PubMed] [Google Scholar]
  15. Kaufman R. J., Murtha P., Ingolia D. E., Yeung C. Y., Kellems R. E. Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3136–3140. doi: 10.1073/pnas.83.10.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Land H., Parada L. F., Weinberg R. A. Cellular oncogenes and multistep carcinogenesis. Science. 1983 Nov 18;222(4625):771–778. doi: 10.1126/science.6356358. [DOI] [PubMed] [Google Scholar]
  18. Mason R. W., Gal S., Gottesman M. M. The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L. Biochem J. 1987 Dec 1;248(2):449–454. doi: 10.1042/bj2480449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mason R. W., Walker J. E., Northrop F. D. The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L. Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line. Biochem J. 1986 Dec 1;240(2):373–377. doi: 10.1042/bj2400373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McCormick M., Gottesman M. E., Gaitanaris G. A., Howard B. H. Cosmid vector systems for genomic DNA cloning. Methods Enzymol. 1987;151:397–405. doi: 10.1016/s0076-6879(87)51031-x. [DOI] [PubMed] [Google Scholar]
  21. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pastan I., Gottesman M. Multiple-drug resistance in human cancer. N Engl J Med. 1987 May 28;316(22):1388–1393. doi: 10.1056/NEJM198705283162207. [DOI] [PubMed] [Google Scholar]
  24. Sahagian G. G., Gottesman M. M. The predominant secreted protein of transformed murine fibroblasts carries the lysosomal mannose 6-phosphate recognition marker. J Biol Chem. 1982 Sep 25;257(18):11145–11150. [PubMed] [Google Scholar]
  25. Scher C. D., Dick R. L., Whipple A. P., Locatell K. L. Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor. Mol Cell Biol. 1983 Jan;3(1):70–81. doi: 10.1128/mcb.3.1.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shen D. W., Cardarelli C., Hwang J., Cornwell M., Richert N., Ishii S., Pastan I., Gottesman M. M. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins. J Biol Chem. 1986 Jun 15;261(17):7762–7770. [PubMed] [Google Scholar]
  27. Shen D. W., Fojo A., Roninson I. B., Chin J. E., Soffir R., Pastan I., Gottesman M. M. Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene. Mol Cell Biol. 1986 Nov;6(11):4039–4045. doi: 10.1128/mcb.6.11.4039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. Stark G. R., Wahl G. M. Gene amplification. Annu Rev Biochem. 1984;53:447–491. doi: 10.1146/annurev.bi.53.070184.002311. [DOI] [PubMed] [Google Scholar]
  30. Stevens T. H., Rothman J. H., Payne G. S., Schekman R. Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol. 1986 May;102(5):1551–1557. doi: 10.1083/jcb.102.5.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Troen B. R., Ascherman D., Atlas D., Gottesman M. M. Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation. J Biol Chem. 1988 Jan 5;263(1):254–261. [PubMed] [Google Scholar]
  32. Troen B. R., Gal S., Gottesman M. M. Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin. Biochem J. 1987 Sep 15;246(3):731–735. doi: 10.1042/bj2460731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ueda K., Cardarelli C., Gottesman M. M., Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A. 1987 May;84(9):3004–3008. doi: 10.1073/pnas.84.9.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES