Nickel-induced heritable alterations in retroviral transforming gene expression

Determination of the mutagenic effects of carcinogenic nickel compounds has been difficult because, like many metals, nickel is poorly or nonmutagenic in procaryotic mutagenicity assays. We attempted to characterize nickel-induced genetic lesions by assessing the effect of nickel chloride on the conditionally defective expression of the v-mos transforming gene in normal rat kidney cells infected with the Murine sarcoma virus mutant ts110 (MuSVts110) retrovirus. MuSVts110 contains an out-of-frame gag gene-mos gene junction that prevents the expression of the v-mos gene at the nonpermissive temperature (39°C). In MuSVts110-infected cells (6m2 cells) grown at 33°C, however, this defect can be suppressed by a splicing event that restores the mos reading frame, allowing the expression of a gag-mos fusion protein which induces the transformed phenotype. The capacity to splice the viral transcript at 33°C, but not at 39°C, is an intrinsic property of the viral RNA. This property allowed us to target the MuSVts110 genome using a positive selection scheme whereby nickel was used to induce genetic changes which resulted in expression of the transformed phenotype at 39°C. We treated 6m2 cells with NiCl₂ and isolated foci consisting of cells which had reverted to the transformed phenotype at 39°C. We found that brief nickel treatment increased the reversion frequency of 6m2 cells grown at 39° C sevenfold over the spontaneous reversion frequency. The nickel-induced revertants displayed the following heritable characteristics: (i) They stably maintained the transformed phenotype at 39°C; (ii) unlike the MuSVts110 TNA in 6m2 cells, the nickel-induced revertant viral RNA could be spliced efficiently at 39°C; (iii) as a consequence of the enhanced accumulation of spliced viral RNA, the nickel-induced revertants produced substantial amounts of the transforming v-mos protein P85(gag-mos) at 39°C; (iv) the nickel-induced revertant P85(gas-mos) serine kinase, like the parental 6m2 P85(gag-mos) kinase, was found to be rapidly inactivated at 39°C; however, in the nickel-induced revertants, overproduction of P85(gag-mos) allowed the transformed state to be maintained; and (v) even though viral RNA processing was much changed, no rearrangements of the viral DNA in the nickel-induced revertant cells were detected by partial restriction analysis.