Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.

Abstract

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C1O and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C1O value in the genomes of cell lines Cdr2OF4, Cdr3OF9, and Cdr200Tl, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr2OF4, CdF3OF9, and Cdr200Ti were equal to 1, 3, and 15 times that measured in Cdr2C1O, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C1O, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in CdF2C10, Cdr3OF9, and Cdr200Tl, but not in Cdr2OF4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr2OF4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.The genomes of mammals encode two closely related metallothioneins (MTs), MT-1 and MT-2, whose expression is induced coordinately by either metals or glucocorticoids (1,13,17). Two different mechanisms have been advanced to account for the coordinate induction of mammalian MT genes. The first has been proposed to explain human MT regulation and is based on differences in the gene dosage and relative inducibilities of the human MT-1 and MT-2 genes. In humans, the MT gene cluster is composed of several functional MT genes and nonfunctional pseudogenes which are located at a number of different chromosomal loci (12,18,19). Mutagenesis and gene transfer experiments have identified regulatory regions upstream from each human MT gene promoter. The sequences upstream from the MT-2 promoter mediate transcription induction by zinc, cadmium, and glucocorticoids (12). In contrast, the sequences upstream from the MT-1 promoter mediate transcription induction by cadmium but are much less responsive to zinc and glucocorticoids (18). Nonetheless, since there are more MT-1 than MT-2 genes, both MT species can be induced coordinately...

Transient response of amplified metallothionein genes in CHO cells to induction by alpha interferon.

“…Effect of interferon treatment on induction of MT genes by zinc. Heavy metals such as zinc and cadmium rapidly induce transcription from MT genes in cultured cells (10,11,15,36). Since interferon also elicits MT expression, we examined whether interferon preexposure influences responsiveness to zinc in Cdr CHO cells.…”

“…Lyophilized human alpha interferon was purchased from Sigma Chemical Co. and reconstituted as suggested by the manufacturer. Plasmids containing MT (15) and alphatubulin (9) cDNA were kindly provided by the Los Alamos Genetics Group and Don Cleveland, respectively.…”

“…Cadmium resistant (Cdr) CHO cell lines which have amplified metallothionein (MT) genes have provided a sensitive system to examine MT gene-inductive processes (15,16). Since MT genes have been shown to be activated during inflammatory and acute-phase reactions (18), the possibility that alpha interferon would transcriptionally activate MT genes in Cdr CHO cells was investigated.…”

Isolation of Mammalian Cell Variants with Enhanced Endogenous Thiol Content at Low Survival Levels Following Irradiation

“…1983, Tobey and Seagrave 1984, Butt et al . 1986, Griffith 1985, Koizumi et al 1985, Kobayashi et al 1985, Mutsubara et al . 1987 in some cases by amplification of the metallothionein genes (Griffith 1985) .…”

“…1986, Griffith 1985, Koizumi et al 1985, Kobayashi et al 1985, Mutsubara et al . 1987 in some cases by amplification of the metallothionein genes (Griffith 1985) . Metallothionein is an efficient scavenger of free radicals (Thornalley and Vasak 1985) and may protect cells (Bakka et al .…”