Differential effects of N-methyl- N′-nitro- N-nitrosoguanidine on constitutive and hormone-inducible gene expression in rat hepatoma cells

Abstract

Previous studies have shown that treatment of rat hepatoma cells (the Fao clone of Reuber H-35 cells) with 500 ng/ml of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) causes a 78% decrease in dexamethasone (DEX)-induced tyrosine aminotransferase (TAT) enzyme activity and a concurrent 75% decline in total steroid-induced TAT steady-state RNA levels. To determine if this inhibition was a specific or more general effect on inducible-gene expression, the effects of MNNG on other genes were examined. MNNG had little effect on total DEX or Cd-induced metallothionein (MT) RNA levels when the cells were treated with 1 μM DEX or 3 μM CdCl 2 for 4 h. In addition, the carcinogen had no effect on the basal level of MT-specific total RNA, nor did it alter the total RNA levels of the α-tubulin gene. Although attempts were made to measure the levels of the glucocorticoid receptor by both biochemical and molecular methods, receptor levels were too low to quantitate accurately. However, the lack of effect of MNNG on steroid-induced MT RNA levels suggests that the inhibitory effect of the carcinogen was not mediated through alterations in glucocorticoid receptor function. MNNG had no effect on cell number or viability, nor did the carcinogen alter the methylation pattern of the TAT gene as determined from MspI/ HpaII digests. The results suggest that MNNG mediates its inhibitory effects by a specific interaction with either the TAT gene itself or some other regulatory factor(s) involved in TAT RNA transcription or stability. This effect was relatively gene specific, since expression of the inducible, specialized liver function TAT gene was inhibited by MNNG but expression of the more ubiquitous and inducible MT gene and the constitutively expressed α-tubulin gene were not.