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. 1995 Nov;147(5):1238–1247.

In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells.

Y Kitadai 1, C D Bucana 1, L M Ellis 1, H Anzai 1, E Tahara 1, I J Fidler 1
PMCID: PMC1869504  PMID: 7485388

Abstract

The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells. The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique. Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression. KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice. The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures. These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.

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Selected References

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

  1. Boag A. H., Young I. D. Increased expression of the 72-kd type IV collagenase in prostatic adenocarcinoma. Demonstration by immunohistochemistry and in situ hybridization. Am J Pathol. 1994 Mar;144(3):585–591. [PMC free article] [PubMed] [Google Scholar]
  2. Bucana C. D., Radinsky R., Dong Z., Sanchez R., Brigati D. J., Fidler I. J. A rapid colorimetric in situ mRNA hybridization technique using hyperbiotinylated oligonucleotide probes for analysis of mdr1 in mouse colon carcinoma cells. J Histochem Cytochem. 1993 Apr;41(4):499–506. doi: 10.1177/41.4.8095509. [DOI] [PubMed] [Google Scholar]
  3. Bussemakers M. J., van Bokhoven A., Mees S. G., Kemler R., Schalken J. A. Molecular cloning and characterization of the human E-cadherin cDNA. Mol Biol Rep. 1993 Feb;17(2):123–128. doi: 10.1007/BF00996219. [DOI] [PubMed] [Google Scholar]
  4. Böhm M., Totzeck B., Birchmeier W., Wieland I. Differences of E-cadherin expression levels and patterns in primary and metastatic human lung cancer. Clin Exp Metastasis. 1994 Jan;12(1):55–62. doi: 10.1007/BF01784334. [DOI] [PubMed] [Google Scholar]
  5. Caruthers M. H., Beaucage S. L., Efcavitch J. W., Fisher E. F., Goldman R. A., deHaseth P. L., Mandecki W., Matteucci M. D., Rosendahl M. S., Stabinsky Y. Chemical synthesis and biological studies on mutated gene-control regions. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):411–418. doi: 10.1101/sqb.1983.047.01.048. [DOI] [PubMed] [Google Scholar]
  6. Collier I. E., Wilhelm S. M., Eisen A. Z., Marmer B. L., Grant G. A., Seltzer J. L., Kronberger A., He C. S., Bauer E. A., Goldberg G. I. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem. 1988 May 15;263(14):6579–6587. [PubMed] [Google Scholar]
  7. Dong Z., Radinsky R., Fan D., Tsan R., Bucana C. D., Wilmanns C., Fidler I. J. Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells. J Natl Cancer Inst. 1994 Jun 15;86(12):913–920. doi: 10.1093/jnci/86.12.913. [DOI] [PubMed] [Google Scholar]
  8. Dorudi S., Sheffield J. P., Poulsom R., Northover J. M., Hart I. R. E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study. Am J Pathol. 1993 Apr;142(4):981–986. [PMC free article] [PubMed] [Google Scholar]
  9. Fabra A., Nakajima M., Bucana C. D., Fidler I. J. Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice. Differentiation. 1992 Dec;52(1):101–110. doi: 10.1111/j.1432-0436.1992.tb00504.x. [DOI] [PubMed] [Google Scholar]
  10. Fan D., Chakrabarty S., Seid C., Bell C. W., Schackert H., Morikawa K., Fidler I. J. Clonal stimulation or inhibition of human colon carcinomas and human renal carcinomas mediated by transforming growth factor-beta 1. Cancer Commun. 1989;1(2):117–125. doi: 10.3727/095535489820875327. [DOI] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Fidler I. J. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990 Oct 1;50(19):6130–6138. [PubMed] [Google Scholar]
  13. Fidler I. J. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis. Cancer Metastasis Rev. 1991 Oct;10(3):229–243. doi: 10.1007/BF00050794. [DOI] [PubMed] [Google Scholar]
  14. Fidler I. J., Radinsky R. Genetic control of cancer metastasis. J Natl Cancer Inst. 1990 Feb 7;82(3):166–168. doi: 10.1093/jnci/82.3.166. [DOI] [PubMed] [Google Scholar]
  15. Fort P., Marty L., Piechaczyk M., el Sabrouty S., Dani C., Jeanteur P., Blanchard J. M. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985 Mar 11;13(5):1431–1442. doi: 10.1093/nar/13.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Galski H., Sullivan M., Willingham M. C., Chin K. V., Gottesman M. M., Pastan I., Merlino G. T. Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia. Mol Cell Biol. 1989 Oct;9(10):4357–4363. doi: 10.1128/mcb.9.10.4357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hart I. R., Easty D. Identification of genes controlling metastatic behaviour. Br J Cancer. 1991 Jan;63(1):9–12. doi: 10.1038/bjc.1991.4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kerbel R. S. Towards an understanding of the molecular basis of the metastatic phenotype. Invasion Metastasis. 1989;9(6):329–337. [PubMed] [Google Scholar]
  19. Levy A. T., Cioce V., Sobel M. E., Garbisa S., Grigioni W. F., Liotta L. A., Stetler-Stevenson W. G. Increased expression of the Mr 72,000 type IV collagenase in human colonic adenocarcinoma. Cancer Res. 1991 Jan 1;51(1):439–444. [PubMed] [Google Scholar]
  20. Matsushima K., Morishita K., Yoshimura T., Lavu S., Kobayashi Y., Lew W., Appella E., Kung H. F., Leonard E. J., Oppenheim J. J. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med. 1988 Jun 1;167(6):1883–1893. doi: 10.1084/jem.167.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morikawa K., Walker S. M., Jessup J. M., Fidler I. J. In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice. Cancer Res. 1988 Apr 1;48(7):1943–1948. [PubMed] [Google Scholar]
  22. Morikawa K., Walker S. M., Nakajima M., Pathak S., Jessup J. M., Fidler I. J. Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res. 1988 Dec 1;48(23):6863–6871. [PubMed] [Google Scholar]
  23. Nagafuchi A., Shirayoshi Y., Okazaki K., Yasuda K., Takeichi M. Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature. 1987 Sep 24;329(6137):341–343. doi: 10.1038/329341a0. [DOI] [PubMed] [Google Scholar]
  24. Naito S., von Eschenbach A. C., Fidler I. J. Different growth pattern and biologic behavior of human renal cell carcinoma implanted into different organs of nude mice. J Natl Cancer Inst. 1987 Feb;78(2):377–385. [PubMed] [Google Scholar]
  25. Nakajima M., Morikawa K., Fabra A., Bucana C. D., Fidler I. J. Influence of organ environment on extracellular matrix degradative activity and metastasis of human colon carcinoma cells. J Natl Cancer Inst. 1990 Dec 19;82(24):1890–1898. doi: 10.1093/jnci/82.24.1890. [DOI] [PubMed] [Google Scholar]
  26. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Poulsom R., Pignatelli M., Stetler-Stevenson W. G., Liotta L. A., Wright P. A., Jeffery R. E., Longcroft J. M., Rogers L., Stamp G. W. Stromal expression of 72 kda type IV collagenase (MMP-2) and TIMP-2 mRNAs in colorectal neoplasia. Am J Pathol. 1992 Aug;141(2):389–396. [PMC free article] [PubMed] [Google Scholar]
  28. Radinsky R., Bucana C. D., Ellis L. M., Sanchez R., Cleary K. R., Brigati D. J., Fidler I. J. A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas. Cancer Res. 1993 Mar 1;53(5):937–943. [PubMed] [Google Scholar]
  29. Radinsky R. Growth factors and their receptors in metastasis. Semin Cancer Biol. 1991 Jun;2(3):169–177. [PubMed] [Google Scholar]
  30. Radinsky R., Kraemer P. M., Raines M. A., Kung H. J., Culp L. A. Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5143–5147. doi: 10.1073/pnas.84.15.5143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Radinsky R. Paracrine growth regulation of human colon carcinoma organ-specific metastasis. Cancer Metastasis Rev. 1993 Sep;12(3-4):345–361. doi: 10.1007/BF00665962. [DOI] [PubMed] [Google Scholar]
  32. Radinsky R., Risin S., Fan D., Dong Z., Bielenberg D., Bucana C. D., Fidler I. J. Level and function of epidermal growth factor receptor predict the metastatic potential of human colon carcinoma cells. Clin Cancer Res. 1995 Jan;1(1):19–31. [PubMed] [Google Scholar]
  33. Reed J. A., Manahan L. J., Park C. S., Brigati D. J. Complete one-hour immunocytochemistry based on capillary action. Biotechniques. 1992 Sep;13(3):434–443. [PubMed] [Google Scholar]
  34. Rogelj S., Weinberg R. A., Fanning P., Klagsbrun M. Basic fibroblast growth factor fused to a signal peptide transforms cells. Nature. 1988 Jan 14;331(6152):173–175. doi: 10.1038/331173a0. [DOI] [PubMed] [Google Scholar]
  35. Singh R. K., Bucana C. D., Gutman M., Fan D., Wilson M. R., Fidler I. J. Organ site-dependent expression of basic fibroblast growth factor in human renal cell carcinoma cells. Am J Pathol. 1994 Aug;145(2):365–374. [PMC free article] [PubMed] [Google Scholar]
  36. Singh R. K., Gutman M., Radinsky R., Bucana C. D., Fidler I. J. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res. 1994 Jun 15;54(12):3242–3247. [PubMed] [Google Scholar]
  37. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  38. Wagner H. E., Toth C. A., Steele G. D., Jr, Thomas P. Metastatic potential of human colon cancer cell lines: relationship to cellular differentiation and carcinoembryonic antigen production. Clin Exp Metastasis. 1992 Jan;10(1):25–31. doi: 10.1007/BF00163573. [DOI] [PubMed] [Google Scholar]
  39. Weinstein R. S., Jakate S. M., Dominguez J. M., Lebovitz M. D., Koukoulis G. K., Kuszak J. R., Klusens L. F., Grogan T. M., Saclarides T. J., Roninson I. B. Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph node metastasis. Cancer Res. 1991 May 15;51(10):2720–2726. [PubMed] [Google Scholar]
  40. Wright J. A., Egan S. E., Greenberg A. H. Genetic regulation of metastatic progression. Anticancer Res. 1990 Sep-Oct;10(5A):1247–1255. [PubMed] [Google Scholar]

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