K-ras ACTIVATION IN RAT RENAL MESENCHYMAL TUMORS INDUCED BY NICKEL SUBSULFIDE

Abstract

Renal mesenchymal tumors were induced in high incidence in male F344 rats by a single intrarenal injection of nickel subsulfide (Ni₃S₂) alone or nickel subsulfide plus iron (Fe°) or magnesium carbonate (MgCarb) (Kasprzak et al., 1990a). The tumors appeared by light microscopy to be comparable in histogenesis to the renal mesenchymal tumors induced by nitrosamines including methyl(methoxymethyl)nitrosamine (DMN-0Me) but were more pleomorphic and had a distinct tendency to metastasize. High-molecular-weight DNA was prepared from the tumors and assayed for transforming activity in NIH 3T3 cells; DNAs from 4 of 16 tumors showed transforming activity. Southern analysis confirmed the presence of the rat K-ras oncogene in the transformed NIH 3T3 clone derived from one of the mesenchymal tumors. Selective oligonucleotide hybridization analysis of polymerase chain reaction (PCR) amplified K-ras gene sequences revealed that 7 of 9 primary tumors induced with N1₃S₂ and Fe° and 1 of 13 primary tumors induced with Ni₃S₂ alone contained exclusively GGT - GTT activating mutations in codon 12. A GGT -. GAT transition was identified in one passaged tumor which had originally contained a GGT - GTT transversion in the primary tumor. The presence of those activating codon 12 point mutations was confirmed by direct sequencing of the PCR amplified K-ras sequences. Sequencing also revealed that there were no other activating mutations at the other codons, 13 and 59-61, that result in activation of the c-K-ras proto-oncogene. These results show that in the rat kidney, Ni₃S₂ carcinogenesis is associated with site-specific activation of a specific cellular proto-oncogene in a fashion consistent with direct interaction of the carcinogen with cellular DNA encoding the target proto-oncogene.