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Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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.

Friend leukemia cells grown in the presence of dimethyl sulfoxide show enhanced erythroid differentiation and hemoglobin synthesis. The effects of dimethyl sulfoxide stimulation are inhibited by BrdU. To determine the effect of BrdU on the amount of globin mRNA present in cells treated and not treated with dimethyl sulfoxide, molecular hybridization between total cell RNA and [ 3 H]DNA complementary to mouse globin mRNA was used. Cells treated with BrdU and dimethyl sulfoxide had 70% less globin mRNA than cells treated with dimethyl sulfoxide alone. The size and base sequence of the residual globin mRNA in the cultures treated with BrdU and dimethyl sulfoxide were unaltered. Cells treated with BrdU alone contained slightly more globin mRNA than did the untreated controls, suggesting that BrdU may have a dual effect in transcription of messenger RNA.

Hemoglobin synthesis in murine Friend virus-induced leukemic cells established in tissue culture has been demonstrated. These cells have exhibited a limited degree of differentiation along the erythroid line throughout their serial passage history. Cloned lines differ in the extent of differentiation as evaluated by benzidine staining and the amount of hemoglobin produced. The hemoglobin present in these cells was identified by its histochemical staining properties, peroxidatic activity, incorporation of 59Fe, absorption spectrum, and characteristic migration pattern during discontinuous electrophoresis on polyacrylamide gels. Three hemoglobin components were detected on 10.0 per cent polyacrylamide gels. The electrophoretic pattern of these three hemoglobins appeared similar to that of hemolysates of adult DBA/2J mice, the strain from which the cells originated.

Tryptic peptides characteristic of mouse hemoglobin α- and diffuse-type β-chains were isolated from dimethylsulfoxide-treated mouse leukemia virus-infected cell cultures. Quantitative estimates of in vitro hemoglobin synthesis by such cultures were obtained by two different methods: by analysis of radioactive amino acid incorporation, and by single-cell radial immunodiffusion assay. The amounts produced, approximately 4 pg/cell/17 hr, reflect a synthetic capacity that is probably sufficient for 32P labeling of hemoglobin messenger RNAs (mRNA) in quantities commensurate with structural characterization of mRNAs by radioautographic methods.

Cells of a cloned line of murine virus-induced 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.

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. The activities of two enzymes in the heme biosynthetic pathway, delta-aminolevulinate dehydratase and uroporphyrinogen-I synthase, were inhibited by 80% and 70%, respectively. Globin mRNA induction was reduced by approximately 90%. This demonstrated that the HC inhibition of globin synthesis occurred at a pretranslational step. The dimethyl sulfoxide-induced single-stranded breaks in DNA, which have been suggested to play a role in Friend leukemia cell differentiation, were reduced in number but not eliminated. HC reduced the dimethyl sulfoxide-stimulation of virus release into the medium by approximately 50%. HC treatment in the absence of dimethyl sulfoxide doubled the production of virus.