Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes

The sensitivity to digestion by DNase I of chromatin containing the alpha- and beta(major)-globin genes and the pattern of DNA methylation near these genes was examined during hexamethylenebisacetamide (HMBA)-mediated erythroid differentiation of murine erythroleukemia cells (MELC). In uninduced and induced cells, the chromatin regions containing the alpha- and beta-(major)-globin genes are more sensitive to digestion by DNase I than is the region containing an immunoglobulin gene (Igalpha) not expressed during erythroid differentiation. However, at low concentrations of DNase I, a 6- to 10-fold increase in site-specific cleavages was generated in chromatin regions near both the alpha- and beta(major)-globin genes in cells induced to differentiate by HMBA. The DNase I hypersensitive site near the beta(major)-globin gene maps to a small region near the 5' terminus of the gene. No detectable change in the pattern of DNA methylation around either the alpha- or beta-globin genes was observed during HMBA-mediated erythroid differentiation. Of the potentially methylated sites assayed and mapped near the beta(major)-globin gene, one site is fully methylated, one is partially methylated, and one is unmethylated both in uninduced and induced cells. Many (but not all) sites assayed near the alpha-globin genes are unmethylated in both uninduced and induced cells. These results show that specific alterations of chromatin structure occur during MELC differentiation and suggest that these changes may not involve alterations in the pattern of DNA methylation.     

Erythroleukemia cells: variants inducible for hemoglobin synthesis without commitment to terminal cell division.

Murine erythroleukemia cells (MELC) are virus-transformed erythroid precursors that appear to be blocked at an erythroid precursor stage comparable to the erythroid colony-forming unit (CFU-e). These cells are useful in examining factors regulating terminal differentiation. Induced MELC are characterized by a coordinated program of gene expression, including commitment to terminal cell division, accumulation of globin mRNAs and corresponding hemoglobins, and accumulation of several other proteins, including the chromatin-associated protein H1(0). Two cloned variant cell lines, DR10 and R1, have been developed from inducer-sensitive DS19 cells by selection for inducer resistance. DR10 and R1 cells fail to display commitment to terminal cell division when cultured with dimethyl sulfoxide (Me2SO), hexamethylene bisacetamide (HMBA), or butyric acid. Both cell lines are induced by all three agents to accumulate H1(0). DR10 cells are resistant to Me2SO-mediated accumulation of hemoglobin but are sensitive to HMBA- or butyric acid-mediated accumulation. R1 cells are resistant to Me2SO- and HMBA-mediated accumulation of hemoglobin but are sensitive to butyric acid-mediated accumulation. Both DR10 and R1 are commitment-negative MELC variants, displaying variable responses to inducers with respect to other features of terminal erythroid cell differentiation.     

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

There are a number of agents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture in HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition of hemin to cultures with HMBA and dexamethasone initiated accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumulation of the chromatin protein IP25; dexamethasone does not inhibit this accumulation. Accumulation of IP25 may be inducer-related, but it is not sufficient to cause expression of terminal erythroid differentiation.     

Relationship between globin mRNA accumulation and commitment to terminal cell differentiation in inducer sensitive and resistant erythroleukemia variants

The relationship between the kinetics of commitment to terminal cell differentiation and the rates of accumulation of globin mRNA has been examined during the induction of erythroid differentiation by polar/apolar chemical inducers in murine erythroleukemia cells (MELC), under conditions of more and less rapid commitment. Two differentiation inducers and three MELC variants have been studied. Hexamethylene bisacetamide (HMBA) initiates more rapid commitment than does dimethylsulfoxide (Me2SO). MELC variant DR10 is resistant to induction by Me2SO and responds sluggishly to HMBA, in comparison with the DS19-Sc9 variant. V3.17, an MELC variant resistant to low concentrations of vincristine, shows increased sensitivity to the inducers and an accelerated rate of commitment to terminal differentiation compared with DS19-Sc9. It is demonstrated that commitment and the actual expression of differentiation, as measured by the accumulation of alpha-, beta maj-, and beta min-globin mRNA, are temporally coordinated functions during induced differentiation of a transformed cell line by exposure to polar/apolar agents.     

Protein kinase C isozymes epsilon and alpha in murine erythroleukemia cells.

Protein kinase C (PKC) has a role in signal transduction during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC). Separation of MELC PKC isozymes by hydroxylapatite chromatography yields a major peak (III) and a minor peak (II) of PKC activity, previously reported to contain the PKC alpha and beta isozymes, respectively. In the present study, we confirm that peak III activity is PKC alpha but show that peak II contains PKC epsilon and little or no PKC beta. Immunoblot analysis with isozyme-specific anti-alpha and anti-epsilon PKC antibodies detected PKC alpha in peak III and PKC epsilon in peak II. Peak III activity was markedly enhanced (up to 20-fold) by phosphatidylserine, diolein, and Ca2+, whereas addition of these cofactors to the reaction mixture stimulated peak II activity only 2- to 4-fold. RNase protection analysis of MELC RNA showed that PKC alpha and PKC epsilon RNAs were in a ratio of approximately 2:1, but PKC beta RNA was barely detectable. Taken together, these data indicate that MELC contain PKC alpha and PKC epsilon but little or no PKC beta.     

Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells.

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.     

Vincristine-resistant erythroleukemia cell line has marked increased sensitivity to hexamethylenebisacetamide-induced differentiation.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A.DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3.17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iii) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBA-mediated DS19 differentiation. We suggest that this V3.17 MEL cell line may express a factor that circumvents HMBA-mediated early events, which prepare the cells for commitment to terminal differentiation.     

Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.

We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.     

Calcium influx in induced differentiation of murine erythroleukemia cells

Murine erythroleukemia cells (MELC) have served as a model for examining the regulation of erythroid differentiation. However, the role of Ca2+ in the signal transduction pathways regulating differentiation remains unclear. To begin to address this uncertainty we have characterized the regulation of cytoplasmic Ca2+ and the possible role of calcium channels during induced differentiation in MELC. MELC can be induced to terminal differentiation using the polar/apolar compound hexamethylene bisacetamide (HMBA). We found that HMBA stimulated Ca2+ influx within 3 to 6 minutes and that Ca2+ entry was required but not sufficient for MELC growth and differentiation. Nifedipine (1 to 10 mumol/L), a calcium channel antagonist, blocked HMBA-induced Ca2+ influx and inhibited differentiation by approximately 60%. Depolarization of the MELC membrane did not induce Ca2+ influx and whole-cell patch-clamp recordings failed to detect a voltage-activated Ca2+ current, suggesting that MELC do not express detectable levels of a functional voltage-dependent calcium channel (VDCC). However, a cDNA probe encoding a portion of the alpha 1 subunit of the cardiac VDCC detected an approximately 8-kb mRNA on Northern blots of total MELC RNA. Taken together, these data show that Ca2+ influx is an early event associated with HMBA-induced differentiation in MELC, blockade of this calcium influx inhibits induced differentiation, and a voltage- insensitive dihydropyridine-sensitive calcium channel may be involved in Ca2+ influx in MELC.