Regulation of a metallothionein-rasT24 fusion gene by zinc results in graded alterations in cell morphology and growth

We constructed fusion genes consisting of the mouse metallothionein I (MT) 5' region and the coding region of either the human H-ras gene (c-rasP3) or a mutated allele (c-rasT24); both ras genes lacked the initial (non-coding) exon and the first 30 bp of the non-coding region of the second exon. Transfection of Rat-1 cells produced foci only with pMT-rasT24, and selection in soft agar yielded clones in which MT-rasT24 expression was zinc-regulatable. In response to increasing concentrations of ZnSO₄, these lines showed increasingly altered morphology (conversion to fusiform or spheroidal morphology), progressively higher maximal cell density, and an increasingly greater fraction of cells in the S+G2+M portion of the cell cycle at high density. MT-rasT24 RNA levels in zinc-responsive lines were increased between 4- and 6-fold by the addition of ZnSO₄ (final concentration = 100 μM) to the medium. Replating cells in the absence of zinc reversed the biological effects and resulted in reduction in MT-rasT24 RNA levels. Thus, graded alterations in phenotype result from increasing levels of MT-rasT24 gene expression.