Influence of Hydrocortisone on the Binding of Nitrosoureas to Nuclear Chromatin Subfractions1

The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 /¿Mhydrocortisone for 22 hr; this resulted in depressed DMA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 rriM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 ¡IM,which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the in creased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin struc ture. Electron microscopic examination of HeLa nuclear mor phology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.