Cells of a cloned line of murine virusinduced erythroleukemia were stimulated to differentiate along the erythroid pathway by dimethyl sulfoxide at concentrations that did not inhibit growth. A rise in the number of benzidine-positive normoblasts was accompanied by increased synthesis of heme and hemoglobin and a decrease in the malignancy of the cells. This action of dimethyl sulfoxide, which was reversible, may represent the derepression of leukemic cells to permit their maturation.The opportunity to explore the possibility that leukemia is a disease resulting from a block in the process of maturation of hematopoietic cells has been provided by established tissue culture lines of murine virus-induced erythroleukemic cells (1-3). These cells, which grow in suspension, have continued to exhibit a limited degree of differentiation along the erythroid line throughout their 4-year serial passage history. Although they synthesize hemoglobin (4, 5), they are malignant as tested by bioassay in syngeneic hosts. They produce virus which, although low in leukemogenic activity, is a highly effective immunizing agent (6).During the course of studies to determine the effect of superinfecting these cells with Friend leukemia virus, dimethyl sulfoxide (DMS0) was added to the medium. DMS0 had been demonstrated to enhance infectivity of both poliovirus RNA (7) and mengovirus RNA (8), as well as transformation by polyoma virus (9). It is also known to stabilize enveloped viruses (10). The wide range of biological activities of DMSO has been described (11). The present report de-scribes an effect of DMSO on the differentiation of established lines of murine virus-induced leukemic cells and illustrates still another property of this compound.In the dose-response experiments initially set up to determine the toxicity of DMSO on the leukemic cell lines, a striking effect on the differentiation of these cells was noted. Of the cells allowed to grow in medium containing 2% DMS0 for 4 days, a majority of the erythroblasts had matured to normoblasts which stained benzidine-positive (B+). The increase in the number of cells maturing along the erythroid series in DMS0-containing medium was accompanied by an increase in the amount of hemoglobin synthesized. MATERIALS AND METHODSThe origin of the cell lines of murine Friend leukemia virusinduced leukemic cells and their clonal derivatives has been described previously (1,2). Methods for maintaining the cell culture, cloning on semisoft agar, and the medium were also detailed in these earlier reports.The present experiments were done on a clone designated 707, in which 1-2% of the cells are B+ (5). Cells were generally seeded at a concentration of 105 per ml (except where otherwise indicated) either in 32-oz. (900 ml) prescription bottles containing 60 ml of medium or in Falcon plastic Petri dishes (60 X 15 mm) containing 10 ml of medium. Dehydrated Eagle's basal medium diluted with Earle's balanced salt solution and supplemented with 15% fetal calf serum was used. The cultures were ...

Treatment of mouse erythroleukemia (MEL) cells with hexamethylene bisacetamide induces a program of erythrodifferentiation, as judged by an increase in the synthesis of globins and other erythroid-specific products. This induction can be inhibited by glucocorticoids, e.g. dexamethasone. All globin and other erythroid-specific genes tested contain GATA response elements (GATA-RE) and can be transactivated by GATA-1, a transcription factor. GATA-1 is highly expressed in erythroid cells, including MEL cells. We noted a glucocorticoid receptor (GR) response element motif near a GATA-RE motif in the promoter region of the mouse beta-major and beta-minor globin genes and about 130 bases away from a GATA-RE in the alpha 1-globin gene promoter and, therefore, investigated the possibility that the dexamethasone-induced inhibition of induced MEL cell differentiation may involve effects of the GR on GATA-1 activity. Evidence obtained from transfection assays and DNA electrophoretic mobility shift assays indicates that the GR binds GATA-1 and interferes with its function before any interaction with DNA, but that the presence of a glucocorticoid response element near a GATA-RE augments the GR effect. The N-terminal 106-amino acid domain of the GR was found to be essential for the effect, possibly by binding to GATA-1. Since GATA-1 is autoregulatory, i.e. it has been shown by others to bind to its own promoter and up-regulate its own transcription, the finding that activated GR can interfere with GATA-1 function may provide an explanation for the inhibition by glucocorticoids of the entire program of erythroid differentiation in MEL cells. That is, by interfering with GATA-1 function, the GR inhibits not only the expression of erythroid structural genes, but may also inhibit the expression of a primary erythroid regulatory gene, GATA-1. It was also shown that the GATA-RE in each of the beta-globin promoters responds to mouse GATA-1 in a functional transfection assay.

Erythrodifferentiation and hemoglobin synthesis in dimethyl sulfoxide-stimulated Friend erythroleukemia cells were inhibited by hydrocortisone (HC) and four other steroids: dexamethasone, deoxycorticosterone, corticosterone, and aldosterone. The effect was specific, because no significant cytotoxicity occurred with any of these compounds at the concentrations that were inhibitory. The mechanism of action of HC was studied in detail. In the absence of dimethyl sulfoxide, it had no effect on hemoglobin levels; but, in the presence of this inducer, the synthesis of heme and globin were each inhibited by approximately 90%. There was no alteration in the synthesis of any major protein other than globin, as determined by gel electrophoresis of cell lysates. Alterations in DNA metabolism (1, 2) and structure (3) are among the many changes that occur in Friend leukemia (FL) cells undergoing dimethyl sulfoxide (Me2SO)-induced erythrodifferentiation (4). The increase in single-stranded breaks in DNA, detected early after Me2SO treatment (5, 6), before globin mRNA appeared (Scher, unpublished), suggested that these breaks might play a role in differentiation in this system as postulated in lens differentiation (7,8) and in transcription (9, 10). Since Me2SO is also known to affect the activity of lysosomes (11,12), the possibility that it was causing the release of specific DNases that might be involved in the production of these scissions was considered. Therefore, a study of the effect of lysosome-stabilizing agents on FL cells was undertaken. Since some compounds known to stabilize lysosomes were found to inhibit erythrodifferentiation of FL cells and others did not, this property did not appear to be correlated with the ability of an agent to inhibit the Me2SO effect.The inhibitory compounds were dexamethasone, hydrocortisone (HG), deoxycorticosterone, corticosterone and aldosterone. The most potent inhibitors were HC and dexamethasone. The mechanism of action of HC, a naturally occurring steroid, was studied further. It markedly inhibited both hemoglobin (Hb) synthesis and globin mRNA levels up to 90% without cytotoxicity, indicating that it acted at a pretranslational step(s). Me2SO-stimulated virus release was also reduced.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. We studied in detail the effect of HG, the most potent of the naturally occurring steroids tested, in an effort to determine the level at which it exerted its inhibitory effect. HC, over a wide range of concentrations, was not cytotoxic and did not affect the cell saturation density (Fig. MATERIALS AND METHODS