Altered oncogenes in UV-transformed C3H 10T1/2 mouse cells: identification of mutated H-ras allele(s).
Ultraviolet (UV) light will transform mammalian cells in culture to a phenotype which is characteristic of in vivo neoplasia. The UV-transformed C3H 10T1/2 mouse cell lines, TU-2 and TU-3, were analysed to determine the molecular mechanisms which may account for their phenotype, and to determine the types of mutations induced by UV light. DNA-transfection assays indicated that the transformed phenotype of TU-2 could not be transferred to non-transformed recipient cells. Therefore, studies were initiated to determine the mutagenic effects of UV light with respect to cellular oncogenes. Northern blot analysis indicated that five of the oncogenes analysed (erb-A, erb-B, mos, myb, and N-ras) were not expressed at detectable levels. The steady-state mRNA levels of fos, K-ras, abl, sis, and src oncogenes were similar in the C3H 10T1/2 and TU-2 cells. The mRNA levels of three oncogenes, raf, myc and H-ras, were 1.5-2.0-fold greater in the TU-2 cells compared to C3H 10T1/2. Southern blot analysis of HpaII restriction digested TU-2 DNA indicated that the H-ras oncogene has undergone methylation changes. More extensive analyses of the H-ras locus in TU-2 demonstrated a deletion of the 3' end of the gene, that may involve two separate mutated alleles. This type of damage is consistent with the lesions associated with sister chromatid exchange. While the H-ras locus in the other UV-transformed line, TU-3, showed methylation changes, there were no large genetic mutations detected by Southern blot analysis. These results suggest that UV-irradiation in vitro induces endogenous DNA damage that includes methylation changes and large genomic alterations. Further analysis will be necessary to determine the extent to which each may be involved in cell transformation.
Thomas
J E
JE
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
Guernsey
D L
DL
D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000528:radiation effects*; D011905:Genes, ras / Q000235:genetics*; D066298:In Vitro Techniques; D008745:Methylation; D051379:Mice; D009154:Mutation; D014466:Ultraviolet Rays