Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 May;9(5):2133–2141. doi: 10.1128/mcb.9.5.2133

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

J H Axelrod 1, R Reich 1, R Miskin 1
PMCID: PMC363007  PMID: 2747645

Abstract

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Full text

PDF
2133

Images in this article

2137Image
on p.2137

Selected References

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

  1. Albini A., Iwamoto Y., Kleinman H. K., Martin G. R., Aaronson S. A., Kozlowski J. M., McEwan R. N. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987 Jun 15;47(12):3239–3245. [PubMed] [Google Scholar]
  2. Bernstein S. C., Weinberg R. A. Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1726–1730. doi: 10.1073/pnas.82.6.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bradley M. O., Kraynak A. R., Storer R. D., Gibbs J. B. Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5277–5281. doi: 10.1073/pnas.83.14.5277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlsen S. A., Ramshaw I. A., Warrington R. C. Involvement of plasminogen activator production with tumor metastasis in a rat model. Cancer Res. 1984 Jul;44(7):3012–3016. [PubMed] [Google Scholar]
  6. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  7. Eisenbach L., Segal S., Feldman M. Proteolytic enzymes in tumor metastasis. I. Plasminogen activator in clones of Lewis lung carcinoma and T10 sarcoma. J Natl Cancer Inst. 1985 Jan;74(1):77–85. [PubMed] [Google Scholar]
  8. Ellis R. W., DeFeo D., Maryak J. M., Young H. A., Shih T. Y., Chang E. H., Lowy D. R., Scolnick E. M. Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus. J Virol. 1980 Nov;36(2):408–420. doi: 10.1128/jvi.36.2.408-420.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
  10. Fidler I. J., Gersten D. M., Hart I. R. The biology of cancer invasion and metastasis. Adv Cancer Res. 1978;28:149–250. doi: 10.1016/s0065-230x(08)60648-x. [DOI] [PubMed] [Google Scholar]
  11. Giraldi T., Sava G., Perissin L., Zorzet S. Proteinases and proteinase inhibition by cytotoxic and antimetastatic drugs in transplantable solid metastasizing tumors in mice. Anticancer Res. 1985 Jul-Aug;5(4):355–359. [PubMed] [Google Scholar]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. Greig R. G., Koestler T. P., Trainer D. L., Corwin S. P., Miles L., Kline T., Sweet R., Yokoyama S., Poste G. Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3698–3701. doi: 10.1073/pnas.82.11.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Günzler W. A., Steffens G. J., Otting F., Kim S. M., Frankus E., Flohé L. The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain. Hoppe Seylers Z Physiol Chem. 1982 Oct;363(10):1155–1165. doi: 10.1515/bchm2.1982.363.2.1155. [DOI] [PubMed] [Google Scholar]
  15. Hagmar B., Ryd W., Erkell L. J. Methods in metastasis research. A critical review. Invasion Metastasis. 1983;3(1):1–21. [PubMed] [Google Scholar]
  16. Hanna N. Expression of metastatic potential of tumor cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity. Int J Cancer. 1980 Nov 15;26(5):675–680. doi: 10.1002/ijc.2910260521. [DOI] [PubMed] [Google Scholar]
  17. Hearing V. J., Law L. W., Corti A., Appella E., Blasi F. Modulation of metastatic potential by cell surface urokinase of murine melanoma cells. Cancer Res. 1988 Mar 1;48(5):1270–1278. [PubMed] [Google Scholar]
  18. Kirchheimer J. C., Pflüger H., Ritschl P., Hienert G., Binder B. R. Plasminogen activator activity in bone metastases of prostatic carcinomas as compared to primary tumors. Invasion Metastasis. 1985;5(6):344–355. [PubMed] [Google Scholar]
  19. Laub O., Rall L. B., Truett M., Shaul Y., Standring D. N., Valenzuela P., Rutter W. J. Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region. J Virol. 1983 Oct;48(1):271–280. doi: 10.1128/jvi.48.1.271-280.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liotta L. A. Tumor invasion and metastases--role of the extracellular matrix: Rhoads Memorial Award lecture. Cancer Res. 1986 Jan;46(1):1–7. [PubMed] [Google Scholar]
  21. Markus G., Camiolo S. M., Kohga S., Madeja J. M., Mittelman A. Plasminogen activator secretion of human tumors in short-term organ culture, including a comparison of primary and metastatic colon tumors. Cancer Res. 1983 Nov;43(11):5517–5525. [PubMed] [Google Scholar]
  22. Markus G. The role of hemostasis and fibrinolysis in the metastatic spread of cancer. Semin Thromb Hemost. 1984 Jan;10(1):61–70. doi: 10.1055/s-2007-1004408. [DOI] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Menzel J., Borth W. Influence of plasma fibronectin on collagen cleavage by collagenase. Coll Relat Res. 1983 May;3(3):217–230. doi: 10.1016/s0174-173x(83)80005-3. [DOI] [PubMed] [Google Scholar]
  25. Mignatti P., Robbins E., Rifkin D. B. Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell. 1986 Nov 21;47(4):487–498. doi: 10.1016/0092-8674(86)90613-6. [DOI] [PubMed] [Google Scholar]
  26. Miskin R., Soreq H. Sensitive autoradiographic quantification of electrophoretically separated proteases. Anal Biochem. 1981 Dec;118(2):252–258. doi: 10.1016/0003-2697(81)90187-1. [DOI] [PubMed] [Google Scholar]
  27. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mullins D. E., Rohrlich S. T. The role of proteinases in cellular invasiveness. Biochim Biophys Acta. 1983 Dec 29;695(3-4):177–214. doi: 10.1016/0304-419x(83)90011-2. [DOI] [PubMed] [Google Scholar]
  29. Nicolson G. L. Cancer metastasis. Organ colonization and the cell-surface properties of malignant cells. Biochim Biophys Acta. 1982 Dec 21;695(2):113–176. doi: 10.1016/0304-419x(82)90020-8. [DOI] [PubMed] [Google Scholar]
  30. Nilsson B., Uhlén M., Josephson S., Gatenbeck S., Philipson L. An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res. 1983 Nov 25;11(22):8019–8030. doi: 10.1093/nar/11.22.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Grady P., Lijnen H. R., Duffy M. J. Multiple forms of plasminogen activator in human breast tumors. Cancer Res. 1985 Dec;45(12 Pt 1):6216–6218. [PubMed] [Google Scholar]
  32. Ossowski L. Plasminogen activator dependent pathways in the dissemination of human tumor cells in the chick embryo. Cell. 1988 Feb 12;52(3):321–328. doi: 10.1016/s0092-8674(88)80025-4. [DOI] [PubMed] [Google Scholar]
  33. Ossowski L., Reich E. Antibodies to plasminogen activator inhibit human tumor metastasis. Cell. 1983 Dec;35(3 Pt 2):611–619. doi: 10.1016/0092-8674(83)90093-4. [DOI] [PubMed] [Google Scholar]
  34. Pennica D., Holmes W. E., Kohr W. J., Harkins R. N., Vehar G. A., Ward C. A., Bennett W. F., Yelverton E., Seeburg P. H., Heyneker H. L. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983 Jan 20;301(5897):214–221. doi: 10.1038/301214a0. [DOI] [PubMed] [Google Scholar]
  35. Ramshaw I. A., Badenoch-Jones P., Grant A., Maxted M., Claudianos C. Enhanced plasminogen activator production by highly metastatic variant cell lines of a rat mammary adenocarcinoma. Invasion Metastasis. 1986;6(3):133–144. [PubMed] [Google Scholar]
  36. Reich R., Thompson E. W., Iwamoto Y., Martin G. R., Deason J. R., Fuller G. C., Miskin R. Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells. Cancer Res. 1988 Jun 15;48(12):3307–3312. [PubMed] [Google Scholar]
  37. Sappino A. P., Busso N., Belin D., Vassalli J. D. Increase of urokinase-type plasminogen activator gene expression in human lung and breast carcinomas. Cancer Res. 1987 Aug 1;47(15):4043–4046. [PubMed] [Google Scholar]
  38. Schirrmacher V. Cancer metastasis: experimental approaches, theoretical concepts, and impacts for treatment strategies. Adv Cancer Res. 1985;43:1–73. doi: 10.1016/s0065-230x(08)60942-2. [DOI] [PubMed] [Google Scholar]
  39. Shih C., Weinberg R. A. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 1982 May;29(1):161–169. doi: 10.1016/0092-8674(82)90100-3. [DOI] [PubMed] [Google Scholar]
  40. Sistonen L., Keski-Oja J., Ulmanen I., Hölttä E., Wikgren B. J., Alitalo K. Dose effects of transfected c-Ha-rasVal 12 oncogene in transformed cell clones. Exp Cell Res. 1987 Feb;168(2):518–530. doi: 10.1016/0014-4827(87)90024-3. [DOI] [PubMed] [Google Scholar]
  41. Skriver L., Larsson L. I., Kielberg V., Nielsen L. S., Andresen P. B., Kristensen P., Danø K. Immunocytochemical localization of urokinase-type plasminogen activator in Lewis lung carcinoma. J Cell Biol. 1984 Aug;99(2):753–757. doi: 10.1083/jcb.99.2.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sloane B. F., Honn K. V., Sadler J. G., Turner W. A., Kimpson J. J., Taylor J. D. Cathepsin B activity in B16 melanoma cells: a possible marker for metastatic potential. Cancer Res. 1982 Mar;42(3):980–986. [PubMed] [Google Scholar]
  43. Soreq H., Miskin R. Plasminogen activator in the rodent brain. Brain Res. 1981 Jul 20;216(2):361–374. doi: 10.1016/0006-8993(81)90138-4. [DOI] [PubMed] [Google Scholar]
  44. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  45. Steffens G. J., Günzler W. A., Otting F., Frankus E., Flohé L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe Seylers Z Physiol Chem. 1982 Sep;363(9):1043–1058. doi: 10.1515/bchm2.1982.363.2.1043. [DOI] [PubMed] [Google Scholar]
  46. Strickland S., Reich E., Sherman M. I. Plasminogen activator in early embryogenesis: enzyme production by trophoblast and parietal endoderm. Cell. 1976 Oct;9(2):231–240. doi: 10.1016/0092-8674(76)90114-8. [DOI] [PubMed] [Google Scholar]
  47. Thorgeirsson U. P., Turpeenniemi-Hujanen T., Williams J. E., Westin E. H., Heilman C. A., Talmadge J. E., Liotta L. A. NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol. 1985 Jan;5(1):259–262. doi: 10.1128/mcb.5.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang B. S., McLoughlin G. A., Richie J. P., Mannick J. A. Correlation of the production of plasminogen activator with tumor metastasis in B16 mouse melanoma cell lines. Cancer Res. 1980 Feb;40(2):288–292. [PubMed] [Google Scholar]
  49. Wexler H. Accurate identification of experimental pulmonary metastases. J Natl Cancer Inst. 1966 Apr;36(4):641–645. doi: 10.1093/jnci/36.4.641. [DOI] [PubMed] [Google Scholar]

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

RESOURCES