Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction. These results strongly support an important role for this natural protein factor in cell differentiation.     

A transfected L-myc gene can substitute for c-myc in blocking murine erythroleukemia differentiation.

We investigated the ability of the proto-oncogene L-myc to substitute for c-myc in blocking murine erythroleukemia differentiation. Murine erythroleukemia cells (line C19) were transfected with recombinant plasmids containing genomic and cDNA fragments of the L-myc gene driven by a Moloney murine leukemia virus long terminal repeat. Clones expressing constitutive high levels of L-myc failed to differentiate in response to the chemical inducer N,N'-hexamethylene bisacetamide (HMBA). The block to differentiation correlated with the level of L-myc expression. Furthermore, transfected clones grown in the presence of inducer for an extended period of time showed an increased level of L-myc expression. These results suggest that functional domains of the c-myc gene involved in differentiation are located in the discrete regions of homology between the c- and L-myc genes.     

Superoxide dismutase induces differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA-induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with liposomes containing D-amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as alpha-tocopherol, butylated hydroxytoluene, or beta-carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.     

Hexamethylene bisacetamide (HMBA) increases thyroglobulin levels in porcine thyroid cells without increasing cyclic-AMP.

The polar planar compound hexamethylene bisacetamide (HMBA) is an inducer of terminal differentiation which has been extensively studied in the murine erythroleukemia cells (MELC). We have tested this compound in normal porcine thyroid cells in primary culture where it either activates or inhibits the major tissue specific functions of these cells: it induces the reorganization of cells into follicles, prevents the loss of thyrotropin sensitivity in monolayer cells, activates cell growth but inhibits their iodide metabolism. In this paper, we demonstrate that HMBA acts on the total thyroglobulin levels measured in cell layers plus media. This specific marker of thyroid tissue is increased by HMBA both in kinetics and in concentration-response experiments. HMBA per se does not increase the total cyclic AMP measured either during the first hours after stimulation or in the following days when compared to controls. As expected, cyclic AMP in the same experiment increased rapidly within minutes after the cells were challenged by TSH (positive control). Altogether, the results show that the drug HMBA mimics thyrotropin effects on thyroglobulin levels measured in porcine thyroid cells in culture. This modulation cannot be explained by an increase in cyclic AMP, indicating that despite similarities between TSH and HMBA effects, the mechanism of the mode of action of these two molecules is very different.     

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.     

Activated macrophages distinguish undifferentiated-tumorigenic from differentiated-nontumorigenic murine erythroleukemia cells

The interaction between macrophages and differentiating cells was examined using murine erythroleukemia cells (MELC). Inflammatory macrophages activated with recombinant murine interferon-gamma (rMuIFN-gamma) and lipopolysaccharide (LPS) first specifically recognized and bound tumorigenic-undifferentiated MELC and then produced their lysis. MELC that were induced to differentiate by a 5-day treatment with 5 mM N,N'-hexamethylene-bis-acetamide (HMBA) accumulated hemoglobin (benzidine positive) and were not recognized by the macrophages. Qualitative examination by light and electron microscopy confirmed the specific nature of the macrophage-MELC interaction. Quantitative assessment showed that the binding was dependent on the temperature and divalent cations and independent of serum components. A 24-h treatment of MELC with HMBA resulted in decreased binding, prior to hemoglobin accumulation and commitment to differentiation. The lack of binding of nontumorigenic-differentiated cells by macrophages was not due to residual HMBA. It thus appears that macrophages can distinguish MELC at different stages of differentiation.     

Catabolites produced by the deacetylation of hexamethylenebisacetamide play a key role in murine erythroleukaemic-cell differentiation.

N-Acetyl-1,6-diaminohexane and 1,6-diaminohexane, formed by deacetylation of the inducer hexamethylenebisacetamide (HMBA), are shown to accumulate rapidly inside murine erythroleukaemic cells. The appearance of these molecules preceded the differentiation-associated changes in intracellular polyamines. A quantitative relationship was observed between the accumulation of these molecules and the changes in intracellular polyamines. In the absence of HMBA, exogenous N-acetyl-1,6-diaminohexane was able not only to cause changes in polyamine biosynthesis, but also to induce the complete differentiation process. These results imply that these catabolites of HMBA are directly responsible for the changes in polyamine biosynthesis and probably also for initiating other events regulatory for the differentiation of these cells.     

Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.     

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [³H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [³H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.     

L'hexaméthylène bisacétamide (hmba) supprime la perte de sensibilité des cellules thyroïdiennes monocouches de porc à la thyrotropine: Hexamethylenebisacetamide (HMBA) prevents the loss of TSH sensitivity in porcine thyroid monolayer cells

The polar coumpound hexamethylenebisacetamide (HMBA) is a differentiating agent in the murine erythroleukemia cell system (MELC). It induces, like dimethylsulfoxide, the commitment to terminal differentiation leading to a recovery in the expression of several genes like the globin gene. This molecule which also induces differentiation in other cellular types is a growth agent for human, ovine and porcine thyroid cells. Forty-eight hours after the onset of culture, porcine thyroid monolayer cells do not respond to thyrotropin (TSH). We demonstrate that a pretreatment from the onset of culture with HMBA of porcine thyroid cells prevents the loss of TSH-sensitivity. The TSH-sensitivity is concentration-dependent in HMBA and leads to the reorganization of cells into follicles, even in the presence of HMBA However, the withdrawal of HMBA when TSH is added is absolutely required to obtain a total recovery in iodide trapping and organification. If HMBA is present during TSH-stimulation, it inhibits iodide trapping partially but iodide organification completely. Cells remain sensitive to TSH for at least 12 days if HMBA treated, and their sensitivity is totally restored after 3, 6 or 9 days of TSH-stimulation. HMBA, which is, like TSH, a growth agent for the thyroid cell and an agent that maintains some of the specialized functions, could be a putative candidate to obtain normal human thyroid cell lines.     

Hybrid polar compounds produce a positive shift in the surface dipole potential of self-assembled phospholipid monolayers

Hybrid polar compounds (HPCs) are powerful inducers of terminal differentiation of various types of tumors, including Friend murine erythroleukemia cells (MELCs). They are known to act synergistically with an increase in the extracellular concentration of cations, which causes a positive shift in the negative value of the ionic surface potential. Two HPCs, hexamethylenebisacetamide (HMBA) and suberoylanilide hydroxamic acid (SAHA), were adsorbed on self-assembled phospholipid monolayers supported on a mercury drop and the shift in the surface dipole potential chi of the lipid film due to their adsorption was estimated from charge measurements. At their optimal concentrations for inducing MELC terminal differentiation (5 mM for HMBA and 2.6 microM for SAHA), these HPCs cause a chi shift of about 15-20 mV, positive toward the hydrocarbon tails, both on neutral phosphatidylcholine films and on negatively or positively charged phosphatidylserine films. This strongly suggests that the nonspecific effect of HPCs of different structure in inducing cancer cells to rescue their differentiation program is related to a positive chi shift on the extracellular side of the cell membrane.     

Benzyl alcohol alters the hemoglobin phenotype of mouse erythroleukemia cells

The hemoglobin minor/hemoglobin major ratio expressed in mouse erythroleukemia (MEL) cells grown in vitro varies according to the differentiation inducer utilized. For example, butyrate and hemin induce higher hemoglobin minor/hemoglobin major ratios than do dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA). Benzyl alcohol in non-toxic concentrations was found to markedly reduce the hemoglobin minor/hemoglobin major ratio and to moderately reduce the total hemoglobin induced by DMSO or HMBA in MEL cells, while only slightly decreasing the ratio induced by hemin or butyrate. The addition of dexamethasone (another and more potent inhibitor of the induction of hemoglobin synthesis than benzyl alcohol) to the media during HMBA induction of differentiation increased the hemoglobin minor/hemoglobin major ratio. This is similar to other "inhibitory" treatments (i.e., treatments that result in sub-optimal hemoglobin production) that have been previously reported. Therefore, although benzyl alcohol and dexamethasone both partly inhibit the induction of total hemoglobin production, they have opposite effects on the induced hemoglobin phenotype: benzyl alcohol decreases the hemoglobin minor/hemoglobin major ratio while dexamethasone increases it. The mechanism(s) of the alteration in the hemoglobin phenotype is unknown as is the mechanism of induction by any of the various inducing agents or of the inhibition of induction by any treatment. However, it appears that if the signal for the induction of hemoglobin minor is sufficiently potent (as it is during butyrate or hemin induction), it cannot be overcome by benzyl alcohol at a "non-toxic" concentration.     

1,6-Diaminohexane contributes to the hexamethylene bisacetamide-induced erythroid differentiation pathway by stimulating Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores and promoting Ca2+ influx

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.     

Murine erythroleukemia cell differentiation: possible involvement of a calcium dependent neutral proteinase

Murine erythroleukemia cells contain a single type of calpain classified, on the basis of its calcium requirement, as a type I proteinase. The enzyme is practically inactive at concentrations of calcium below 10 microM and expresses maximal activity in the presence of 0.12-0.15 mM Ca2+. The affinity for Ca2+ cannot be reduced by exposure of the enzyme to conditions known to promote autoproteolysis of calpain. Expression of catalytic activity at lower concentrations of Ca2+, is promoted by the interaction with phospholipid vesicles or plasma membranes. Conditions that promote activation of calpain, induce also the self-inactivation of the enzyme. During terminal differentiation of murine erythroleukemia cells induced by HMBA, the intracellular level of calpain activity undergoes significative reduction. Similar decrease in calpain activity progressively occurs during the loss of sensitivity to HMBA as a result of density growth arrest.     

Changes in p34cdc2 kinase activity and cyclin A during induced differentiation of murine erythroleukemia cells.

Hexamethylene bisacetamide (HMBA)-induced murine erythroleukemia (MELC) differentiation is characterized by a prolongation of the initial G1 which follows passage through S phase in the presence of inducer. Commitment to terminal cell division is first detected in a portion of the cell population during this prolonged G1. HMBA-induced commitment is stochastic. This study has examined changes in two known cell cycle regulators, p34cdc2 and cyclin A, in cycle-synchronized MELC in the absence and presence of HMBA. Histone H1 kinase activity of p34cdc2, and the levels of CDC2Mm mRNA, 1.8-kilobase mRNA of cyclin A, and cyclin A protein changed during cell cycle progression in MELC, and all of them were suppressed during G1. The suppression of the H1 kinase activity and cyclin A expression continued through the prolonged G1 in MELC cultured with HMBA, whereas p34cdc2 protein level did not vary through the cell cycle in MELC cultured without or with inducer. Phosphorylation of p34cdc2 in uninduced MELC gradually increased as cells progressed from G1 to S. In induced MELC, an increase in phosphorylation of p34cdc2 occurred during the prolonged G1, and prior to the exit of the bulk of the cells from G1 to S. These results suggest that in HMBA-induced MELC, p34cdc2 phosphorylation per se is not a limiting factor in determining G1 to S progression. The persistent suppression of cyclin A expression and histone H1 kinase activity may play a role in HMBA-induced commitment to terminal differentiation.     

In vitro phosphorylation of lamin B by protein kinase C in friend erythroleukemia. Effect of chemically induced differentiation

Nuclear matrix isolated from murine erythroleukemia cells (Friend cells) has been phosphorylated with gamma 32P-ATP and purified protein kinase C in order to identify specific nuclear substrates for the enzyme. HMBA has been employed to induce the cell to differentiate and to compare the changes of phosphorylation profile after erythroid differentiation. Lamin B has been found to be hyperphosphorylated by rat brain PK-C in nuclear matrix purified from uninduced cells. This difference characterizes the cells from 14 to 72 hrs of HMBA treatment and indicates that the ability of lamin B to be phosphorylated by PK-C is linked to the differentiated state. The involvement of PK-C in lamin phosphorylation might represent an early step of the signalling pathway utilized by erythroid differentiating agents to target the cell nucleus.     

A rise and fall in 1,2-diacylglycerol content signal hexamethylene bisacetamide-induced erythropoiesis.

Previous studies have suggested a role for protein kinase C (PKC) during induction of murine erythroleukemia cell (MELC) differentiation by hexamethylene bisacetamide (HMBA) (Melloni, E., Pontremoli, S., Viotti, P. L., Patrone, M., Marks, P. A., and Rifkind, R. A. (1989) J. Biol. Chem. 264, 18414-18418). The present studies assess the effect of HMBA on the content of 1,2-diacylglycerol (DG), the physiologic activator of PKC, in MELC variants. Exposure of parental Sc9 cells to HMBA induced a rapid rise and fall in DG content. The DG level increased within seconds from 225 pmol.10(6) cells-1 to a maximum of 305 pmol.10(6) cells-1 at 5 min. Thereafter, DG content fell reaching control levels at 30 min and 46% of control at 4 h. Similar DG elevations were detected in HMBA-resistant, phorbol ester-resistant, and vincristine-resistant MELC lines. Early DG elevation was followed by the characteristic rapid fall in both the phorbol ester-resistant and vincristine-resistant lines, both of which differentiate rapidly in response to HMBA. In contrast, in an HMBA-resistant MELC the DG level failed to fall for at least 10 h. Selection of HMBA-resistant cells for vincristine resistance restores both HMBA sensitivity and the rapid fall in DG content. Addition of a synthetic DG, 1-oleyl-2-acetyl glycerol (OAG), along with HMBA and every 2 h for the next 48 h blocked differentiation, as measured by accumulation of benzidine-reactive cells or by the commitment assay in methyl-cellulose. However, if addition of OAG was delayed for just a few minutes, until endogenous DG levels began to fall, differentiation was no longer inhibited. Rapid elevation of DG content is the earliest reported event during HMBA action and a subsequent fall in the DG content appears to be a critical step in the process of commitment to terminal differentiation.