Spontaneous Ha‐ras Gene Activation in Cultured Primary Murine Keratinocytes: Consequences of Ha‐ras Gene Activation in Malignant Conversion and Malignant Progression

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Abstract
The activation of the c‐Ha‐ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2–3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c‐Ha‐ras gene with an A‐>T transversion in codon 61. Fibroblast‐conditioned low Ca2+ medium prevented cell death, focus formation, c‐Ha‐ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c‐Ha‐ras gene of keratinocytes. When a v‐Ha‐ras gene was introduced into normal keratinocytes by a replication‐defective retrovirus, the recipient cells produced papillo‐mas in vivo, and after 2 mo, 60% of the tumors converted to squamous cell carcinomas. None of the 22 converted tumors had an endogenous c‐Ha‐ras gene mutation at codon 61. However, the A‐>T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast‐conditioned medium. Cells with both an exogenous v‐Ha‐ras and an activated c‐Ha61‐ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v‐Ha‐ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v‐Ha‐ras gene was introduced into papilloma cells with a chemically induced endogenous c‐Ha61‐ras gene mutation. These results suggest that mutation in the c‐Ha‐ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.