Oncogene-mediated transformation of fetal rat colon in vitro.
Short-term maintenance of fetal rat colonic tissue in vitro has been demonstrated using a collagen matrix organ culture system. The introduction of single (v-myc, v-rasH, v-src) oncogenes or combinations of oncogenes (v-myc/rasH, v-myc/src) into normal colon mucosal elements was established using retroviral vectors, resulting in enhanced proliferation and migration of epithelial cells from the lumen of tissue implants. Expression of a single oncogene in normal colon epithelium did not result in the establishment of cell lines. In contrast, expression of cooperating oncogenic elements resulted in cell lines in greater than 80% of experiments, revealing different morphological characteristics dependent upon the oncogene combination used. Confirmation of the expression of viral transcripts was determined using Northern blot analysis and viral oncoprotein expression using Western blot analysis (p21) and an immunoprecipitation kinase assay (src). Expression of keratin filaments was lost following passaging of cell lines but could be induced by the myc/ras transformants by growth on Rat-1 feeder layers. This induction phenomenon was not observed with myc/src lines, and although these expressed high levels of sucrase isomaltase the epithelial origin of these cells is unclear. Karyotypic analysis performed on three myc/ras-transformed cell lines revealed a normal chromosome complement associated with transformation. In this report we describe a novel in vitro transformation system for normal rat colonic epithelium mediated by the introduction of oncogene elements using different retroviral vector constructs. The potential to generate cell lines representing different stages of neoplastic progression using relevant genetic components presents significant advantages for the study of cellular and molecular interactions underlying colon neoplastic progression.
Pories
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Laboratory of Cancer Biology, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Jaros
K
K
Steele
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G
Pauley
A
A
Summerhayes
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IC
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002352:Carrier Proteins / Q000096:biosynthesis; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003106:Colon / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D016259:Genes, myc / Q000502:physiology; D011905:Genes, ras / Q000502:physiology; D016391:Genes, src / Q000502:physiology; D007621:Karyotyping; D007633:Keratins / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008840:Microfilament Proteins / Q000096:biosynthesis; D009857:Oncogenes / Q000502:physiology*; D009924:Organ Culture Techniques; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis; D012313:RNA / Q000032:analysis; D051381:Rats; D011920:Rats, Inbred WF; D013394:Sucrase-Isomaltase Complex / Q000096:biosynthesis; D019061:src-Family Kinases
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D016271:Proto-Oncogene Proteins c-myc; C026742:plastin; C026615:villin; D012313:RNA; D007633:Keratins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; D013394:Sucrase-Isomaltase Complex; D016283:Proto-Oncogene Proteins p21(ras)