William Scher

The structural basis for steroid modulation of DMSO-stimulated erythrodifferentiation

Abstract The modulatory effect of 47 steroids on the DMSO stimulation of hemoglobin production in murine erythroleukemia cells was determined. The steroids capable of optimal inhibition of the DMSO effect most severely were glucocorticoids. Three steroids-halcinonide, flumethasone pivalate and flucloronide-were found to be active inhibitors at concentrations even lower than those noted for dexamethasone. Several steroids were noted to be capable of blocking the dexamethasone inhibition. Of these anti-inhibitors, fluoxymesterone and 17α-hydroxyprogesterone were the most active. The inhibitory and anti-inhibitory steroids were found to be the same steroid agonists or antagonists noted in other systems which are known to bind to glucocorticoid receptors. Therefore, it is likely that the activity of these steroids in erythroleukemia cell differentiation is mediated by typical glucocorticoid receptors. It was also shown that steroids strongly inhibit the DMSO effect only when present during DMSO stimulation of globin mRNA levels. This was further evidence that steroids were not acting at the level of translation. Since the same glucocorticoids that inhibit erythroid maturation in the mouse cells studied here also induce mouse lymphocytolysis and granulocytic differentiation, it is suggested that glucocorticoids may play an overall regulatory role in vivo in the determination of the numbers of mature blood cells of different types functioning in the organism. This type of corticosteroid action may occur in addition to any steroidal effects at earlier stages in hematopoiesis.

DNA LIGASE ACTIVITY IN EXTRACTS OF MOUSE ERYTHROLEUKEMIA (MEL) CELLS DURING DIFFERENTIATION

Treatment with dimethyl sulfoxide (DMSO) induces some stages of erythroid differentiation in MEL cells. The differentiating cells produce hemoglobin (Hb) and become limited in their proliferative capacity. Nuclear condensation occurs (see Reuben et al.l for review). After 4 days in culture in the absence and in the presence of 1.8% (vol/vol) DMSO, DNA ligase levels were shown to be lower in DMSO-treated MEL cells than in untreated MEL cells? To further characterize this effect, DNA ligase and DNAase activities were determined in cell-free extracts prepared from MEL cells grown from 1 to 5 days in the absence and in the presence of DMSO. Hb-containing cells were detectable after 67 hr of growth in the presence of DMSO and by 1 1 3 hr the percentage of such cells in the DMSO-treated cultures had risen to over 85%. In contrast, the percentage of Hb-containing cells in untreated cultures remained at less than 1 % . DNA ligase activity was measured under conditions that inhibited all major competing DNAase activities in the extracts. The specific activity of DNA ligase in extracts from DMSO-treated cells was 167, 95, 22, 12, and <4% of the activity in extracts from untreated cells after 19, 43, 67, 95, and 113 hr of DMSO treatment, respectively. When extracts from untreated and DMSOtreated cells were mixed, DNA ligase activities in the mixtures were additive and no evidence of inhibitors in the extracts from the DMSO-treated cells was noted. DNAase activity was determined in extracts that were first dialyzed to eliminate ATP and therefore DNA ligase activity. The major DNAase activity in MEL cell extracts was inhibited by G-actin and was, therefore, presumed to be DNAase I-like. Unlike the results seen with DNA ligase, the specific activity of this DNAase did not decrease during 95 hr of DMSO treatment, but rather increased slightly. In cell line DR-10, a variant cell line that is resistant to DMSO induction of differentiation, these DMSO-induced changes were not seen. In addition,