Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Correlation between Ha-ras gene amplification and spontaneous metastasis in NIH 3T3 cells transfected with genomic DNA from human skin cancers

Abstract

Our previous studies have shown that DNA from some human skin cancers contained activated Ha-ras oncogenes capable of inducing tumorigenic transformation when introduced into NIH 3T3 cells by DNA-mediated gene transfer. In addition, we found that NIH 3T3 cells transfected with DNA from one of the human skin cancers not only induced s.c. tumors at the site of injection but also metastasized spontaneously to the lungs in 100 per cent of nude mice injected. In this present study we examined the relationship between Ha-ras oncogene amplification and metastatic potential in tumors induced by various human skin cancer DNA-transfectants. Total cellular RNA was extracted from nude mouse tumor cell lines and analyzed by northern blot hybridization to a32P-labeled, nick-translated Ha-ras probe. The metastatic potential of nude mouse tumor cell lines was assessed by their ability to form lung colonies after i.v. or s.c. injection. It was found that only the tumors expressing high levels of Ha-ras gene transcripts induced spontaneous metastasis after s.c. injection. There appeared to be little correlation between the level of Ha-ras oncogene amplification and experimental metastasis. These results suggest that amplification and overexpression of Ha-ras oncogene may play a role in the escape of cells from the primary tumor rather than in the ability of cells to survive in the circulatory system and colonize secondary sites.

This is a preview of subscription content, log in to check access.

Abbreviations

HBSS:

Hank's balanced salt solution

SSC:

Standard saline citrate

SDS:

Sodium dodecyl sulfate

s.c.:

Subcutaneous

i.v.:

Intravenous

References

  1. [1]

    Ananthaswamy, H. N., Price, J. E., Goldberg, L. H., andBales, E. S., 1988, Simultaneous transfer of tumorigenic and metastatic phenotypes by transfection with genomic DNA from a human cutaneous squamous cell carcinoma.Journal of Cellular Biochemistry,36, 137–146.

  2. [2]

    Ananthaswamy, H. N., Price, J. E., Goldberg, L. H., andBales, E. S., 1988, Detection and identification of activated oncogenes in human skin cancers occurring on sun exposed sites.Cancer Research,48, 3341–3346.

  3. [3]

    Balmain, A., andPragnell, I. B., 1983, Mouse skin carcinomas inducedin vivo by chemical carcinogens have a transforming Harvey-ras oncogene.Nature (London),303, 72–74.

  4. [4]

    Balmain, A., Ramsden, H., Bowden, G. T., andSmith, J., 1984, Activation of the mouse cellular Harvey-ras gene in chemically induced benign papillomas.Nature (London),307, 658–660.

  5. [5]

    Bernstein, S. C., andWeinberg, R. A., 1985, Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.Proceedings of the National Academy of Sciences of the U.S.A.,82, 1726–1730.

  6. [6]

    Bishop, M. J., 1983, Cellular oncogenes and retroviruses.Annual Review of Biochemistry,52, 301–354.

  7. [7]

    Bondy, G. P., Wilson, S., andChambers, A. F., 1985, Experimental metastatic ability of H-ras-transformed NIH 3T3 cells.Cancer Research,45, 6005–6009.

  8. [8]

    Bradley, M. O., Kraynak, A. R., Storer, R. D., andGibbs, J. B., 1986, Experimental metastasis in nude mice of NIH 3T3 cells containing variousras genes.Proceedings of the National Academy of Sciences of the U.S.A.,83, 5277–5281.

  9. [9]

    Chirgwin, J. M., Przbyla, A. E., McDonald, R. J., andRutter, W. J., 1979, Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry,18, 5294–5299.

  10. [10]

    Collard, J. G., Schijven, J. F., andRoss, E., 1987, Invasive and metastatic potential induced by ras-transfection into mouse BW5147 T-lymphoma cells.Cancer Research,47, 754–759.

  11. [11]

    Dandekar, S., Sukumar, S., Zarbl, H., Young, J. T., andCardiff, R. D., 1986, Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthraceneinduced mouse mammary tumors.Molecular and Cellular Biology,6, 4104–4108.

  12. [12]

    Der, C., Krontiris, T., andCooper, G., 1982, Transforming genes of human bladder and lung carcinoma cell lines are homologous to theras genes of Harvey and Kirsten sarcoma viruses.Proceedings of the National Academy of Sciences of the U.S.A.,79, 3637–3640.

  13. [13]

    Egan, S. E., McClarty, G. A., Jarolim, L., Wright, J. A., Spiro, I., Hager, G., andGreenberg, A. H., 1987, Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.Molecular and Cellular Biology,7, 830–837.

  14. [14]

    Egan, S. E., Wright, J. A., Jarolim, L., Yanagihara, K., Bassin, R. H., andGreenberg, A. H., 1987, Transformation by oncogenes encoding protein kinases induces the metastatic phenotype.Science,238, 202–206.

  15. [15]

    Eva, A., andAaronson, S. A., 1983, Frequent activation of c-kis as a transforming gene in fibrosarcomas induced by methylcholanthrene.Science,220, 955–956.

  16. [16]

    Fasano, O., Birnbaum, D., Edlund, E., Fogh, J., andWigler, M., 1984, New human transforming genes detected by a tumorigenicity assay.Molecular and Cellular Biology,4, 1695–1705.

  17. [17]

    Fidler, I. J., Gersten, D. M., andHart, I. R., 1978, The biology of cancer invasion and metastasis.Advances in Cancer Research,28, 149–250.

  18. [18]

    Gallick, G. E., Kurzrock, R., Kloetzer, W. S., Arlinghaus, R. B., andGutterman, J. U., 1985, Expression of p21ras in fresh primary and metastatic human colorectal tumors.Proceedings of the National Academy of Sciences of the U.S.A.,82, 1795–1799.

  19. [19]

    Goldfarb, M., Shimizu, K., Perucho, M., andWigler, M., 1982, Isolation and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells.Nature (London),296, 404–409.

  20. [20]

    Greig, R. G., Koestler, T. P., Trainer, D. L., Corwin, S. P., Miles, L., Kline, T., Sweet, R., Yokoymama, S., andPoste, G., 1985, Tumorigenic and metastatic properties of ‘normal’ and ras-transfected NIH/3T3 cells.Proceedings of the National Academy of Sciences of the U.S.A.,82, 3698–3701.

  21. [21]

    Lane, M. A., Sainten, A., andCooper, G. M., 1981, Activation of related transforming genes in mouse and human mammary carcinomas.Proceedings of the National Academy of Sciences of the U.S.A.,78, 5185–5189.

  22. [22]

    Lundy, J., Grimson, R., Mishriki, Y., Chao, S., Oravez, S., Fromowitz, F., andViola, M. V., 1986, Elevatedras expression correlates with lymph node metastases in breast cancer patients.Journal of Clinical Oncology,4, 1321–1325.

  23. [23]

    Maniatis, T., Fritsch, E. F., andSambrook, J., 1982,Molecular Cloning. A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory), p. 86.

  24. [24]

    Muschel, R. J., Williams, J. E., Lowy, D. R., andLiotta, L. A., 1985, Harveyras induction of metastatic potential depends upon oncogene activation and the type of recipient cell.American Journal of Pathology,1, 1–8.

  25. [25]

    Ohuchi, N., Thor, A., Page, D. L., Horan Land, P., Halter, S. A., andScholm, J., 1986, Expression of the 21,000 molecular weightras protein in a spectrum of benign and malignant human mammary tissues.Cancer Research,46, 2511–2519.

  26. [26]

    Parada, L. F., Tabin, C. J., Shih, C., andWeinberg, R. A., 1982, Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virusras gene.Nature (London),297, 474–478.

  27. [27]

    Parada, L. F., andWeinberg, R. A., 1983, Presence of a Kirsten murine sarcoma virusras oncogene in cells transformed by 3-methylcholanthrene.Molecular and Cellular Biology,3, 2298–2301.

  28. [28]

    Pellicer, A., Wigler, M., Axel, R., andSilverstein, S., 1978, The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.Cell,27, 467–476.

  29. [29]

    Perucho, M., Goldfarb, M., Shimizu, K., Lama, C., Fogh, J., andWigler, M., 1981, Human-tumor-derived cell lines contain common and different transforming genes.Cell,27, 467–476.

  30. [30]

    Poste, G., Doll, J., Hart, I. R., andFidler, I. J., 1980,In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Research,40, 1636–1644.

  31. [31]

    Pozzattti, R., Muschell, R., Williams, J., Padmanabhan, R., Howard, B., Liotta, L., andKhoury, G., 1986, Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials.Science,232, 223–228.

  32. [32]

    Pulciani, S., Santos, E., Lauver, A. V., Long, L. K., Aaronson, S. A., andBarbacid, M., 1982, Oncogenes in solid tumours.Nature (London),300, 539–542.

  33. [33]

    Pulciani, S., Santos, E., Lauver, A. V., Long, L. K., Robbins, K. C., andBarbacid, M., 1982, Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.Proceedings of the National Academy of Sciences of the U.S.A.,79, 2845–2849.

  34. [34]

    Pulciani, S., Santos, E., Long, L. K., Sorrentino, V., andBarbacid, M., 1985,ras gene amplification and malignant transformation.Molecular and Cellular Biology,5, 2836–2841.

  35. [35]

    Santos, E., Tronick, S. R., Aaronson, S. A., Pulciani, S., andBarbacid, M., 1982, T24 human bladder carcinoma oncogene is an activated form of the normal human homologue of BALB- and Harvey-MSV transforming genes.Nature (London),298, 343–347.

  36. [36]

    Santos, E., Reddy, E. P., Pulciani, E., Feldman, R. J., andBarbacid, M., 1983, Spontaneous activation of a human proto-oncogene.Proceedings of the National Academy of Sciences of the U.S.A.,80, 4679–4683.

  37. [37]

    Shimizu, K., Goldfarb, M., Suard, Y., Perucho, M., Li, Y., Kamata, T., Feramisco, J., Stavnezer, E., Fogh, J., andWigler, M., 1983, Three human transforming genes are related to the viralras oncogenes.Proceedings of the National Academy of Sciences of the U.S.A.,80, 2112–2116.

  38. [38]

    Southern, E. M., 1975, Detection of specific sequences among DNA fragments separated by gel electrophoresis.Journal of Molecular Biology,98, 503–517.

  39. [39]

    Tahara, E., Yasui, W., Taniyama, K., Ochial, A., Yamamoto, T., Nakajo, S., andYamamoto, M., 1986, Ha-ras oncogene product in human gastric carcinoma: correlation with invasiveness, metastasis or prognosis.Japan Journal of Cancer Research (Gann),77, 517–522.

  40. [40]

    Tainsky, M. A., Cooper, C. S., Giovanella, B. C., andVande Woude, G. F., 1984, An activatedras gene: detected in late but not early passage human PA1 teratocarcinoma cells.Science,225, 643–645.

  41. [41]

    Tarin, D., 1987, Studies of the genetic basis of tumor metastasis.Journal of Cellular Biochemistry, Suppl.11D, 86.

  42. [42]

    Thor, A., Horan Land, P., Wunderlich, D., Caruso, A., Muraro, R., andSchlom, J., 1984, Monoclonal antibodies define differentialras gene expression in malignant and benign colonic diseases.Nature (London),311, 562–565.

  43. [43]

    Thorgeirsson, U. P., Turpeenniemi-Hujanen, T., Williams, J. E., Westin, E. H., Heilman, C. A., Talmadge, J. E., andLiotta, L. A., 1985, NIH/3T3 cells transfected with human tumor DNA containing activatedras oncogenes express the metastatic phenotype in nude mice.Molecular and Cellular Biology,5, 259–262.

  44. [44]

    Vousden, K. H., andMarshall, C. J., 1984, Three different activatedras genes in mouse tumors; evidence for oncogene activation during progression of a mouse lymphoma.European Molecular Biology Organization Journal,3, 913–917.

  45. [45]

    Vousden, K. H., Eccles, S. A., Purvies, H., andMarshall, C. J., 1986, Enhanced spontaneous metastasis of mouse carcinoma cells transfected with an activated c-Ha-ras-1 gene.International Journal of Cancer,37, 425–433.

Download references

Author information

Correspondence to Honnavara N. Ananthaswamy or Janet E. Price.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ananthaswamy, H.N., Price, J.E., Tainsky, M.A. et al. Correlation between Ha-ras gene amplification and spontaneous metastasis in NIH 3T3 cells transfected with genomic DNA from human skin cancers. Clin Exp Metast 7, 301–313 (1989). https://doi.org/10.1007/BF01753682

Download citation

Keywords

  • Nude Mouse
  • Gene Transcript
  • Northern Blot
  • Skin Cancer
  • Gene Amplification