Protein kinase C activity and hexamethylenebisacetamide-induced erythroleukemia cell differentiation.

Hexamethylenebisacetamide (HMBA) is a potent inducer of murine erythroleukemia (MEL) cell differentiation. The mechanism of action of HMBA is not known. In this study we provide evidence that protein kinase C has a role in inducer-mediated MEL cell differentiation: (i) HMBA induces the formation of a soluble, proteolytically activated form of protein kinase C that is catalytically active in the absence of Ca2+ and phospholipid; (ii) the protease inhibitor leupeptin blocks formation of this activated form of the kinase and inhibits HMBA-induced MEL cell hemoglobin accumulation; (iii) phorbol 12-myristate 13-acetate (PMA) inhibits HMBA-induced MEL differentiation and causes depletion of total protein kinase C activity; (iv) MEL cells depleted in protein kinase C activity by culture with PMA are resistant to induction by HMBA; (v) upon removal of PMA, restoration of MEL cell sensitivity to HMBA is correlated with reaccumulation of protein kinase C activity; and (vi) MEL cells grown to density arrest are both depleted of protein kinase C activity and resistant to HMBA. Together, these results suggest that HMBA-mediated MEL cell differentiation involves a protein kinase C-related mechanism and the proteolytically activated form of the kinase, which does not require Ca2+ or phospholipid for its catalytic activity.     

Inducing differentiation of transformed cells with hybrid polar compounds: a cell cycle-dependent process.

Transformed cells do not necessarily lose their capacity to differentiate. Various agents can induce many types of neoplastic cells to terminal differentiation. Among such inducers, a particularly potent group consists of hybrid polar compounds; hexamethylene bisacetamide (HMBA) is the prototype of this group. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. This review focuses on HMBA-induced modulation of factors regulating G1-to-S phase progression, including a decrease in the G1 cyclin-dependent kinase cdk4, associated with inhibition of phosphorylation of the retinoblastoma protein pRB and possibly other related proteins that, in turn, sequester factors required for initiation of DNA synthesis; this provides a possible mechanism for HMBA-induced terminal cell division. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment will also be reviewed.     

Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12–48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.     

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.     

Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.     

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.     

Changes in gene expression associated with induced differentiation of erythroleukemia: protooncogenes, globin genes, and cell division.

Hexamethylenebisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC) is a multistep process involving an early latent period during which a number of metabolic changes have been detected, but the cells are not yet committed irreversibly to differentiate. Commitment is defined as the capacity of MELC to go on to express the program of terminal cell division and gene expression (such as the accumulation of globin mRNA) upon removal of the HMBA from the culture. In the presence of HMBA, a small proportion of MELC are committed by 10-12 hr and greater than 90% by 48-60 hr. The present study shows that, during the initial 4 hr of culture, HMBA causes a marked decrease in c-myb and c-myc and an increase in c-fos mRNA levels. With continued culture, the decrease in c-myb and the increase in c-fos mRNA persists, while c-myc mRNA returns to control levels before the time that MELC begin to show irreversible differentiation. Dexamethasone, which blocks expression of HMBA-induced MELC differentiation, does not alter the early pattern of changes in protooncogene mRNA nor the sustained elevation of c-fos, but it does inhibit the continued suppression of c-myb allowing c-myb to return toward control levels. Hemin, which induces MELC to accumulate globins but does not initiate commitment to terminal cell division, does not alter these protooncogene mRNA levels. These studies suggest that, although the early decrease in c-myb and c-myc and increase in c-fos mRNAs may be involved in the multistep events leading to differentiation, the continued suppression of c-myb is critical for HMBA-induced MELC commitment to terminal cell division.     

Hexamethylenebisacetamide-induced erythroleukemia cell differentiation involves modulation of events required for cell cycle progression through G1.

Hexamethylenebisacetamide (HMBA), a potent inducer of differentiation of transformed cells such as murine erythroleukemia cells, causes a prolongation of the G1 phase of the cell cycle during which commitment to terminal differentiation is first detected. Removal of HMBA prior to the G1 phase aborts commitment. To further define the relationship between the G1 phase and commitment to differentiation, we used two inhibitors of cell cycle progression: aphidicolin, which blocks cells at the G1/S interphase, and deferoxamine, which blocks cells at an earlier stage during G1. HMBA-induced prolongation of G1 is associated with the accumulation of underphosphorylated retinoblastoma protein, decrease in cyclin A protein levels, and commitment to differentiation. G1 arrest of murine erythroleukemia cells induced by aphidicolin or deferoxamine is not associated with accumulation of under-phosphorylated retinoblastoma protein, suppression of cyclin A protein, or commitment of cells to terminal differentiation. Neither of the cell cycle inhibitors alters the effect of HMBA in inducing the G1-associated changes or commitment to differentiation. Taken together, the present findings indicate that the site of action of HMBA which leads to commitment is in a stage of the G1 phase prior to the point of cell cycle block caused by deferoxamine or aphidicolin. HMBA appears to cause cell differentiation with suppression of cell cycle progression by an action that affects events required for cell progression through G1, including accumulation of underphosphorylated retinoblastoma protein and changes in regulation of cyclin levels.     

A novel system to identify Myb target promoters in friend murine erythroleukemia cells

Friend murine erythroleukemia (MEL) cells provide an early erythroid precursor model that can be induced to terminally differentiate in cell culture and has been used to study erythroid differentiation as well as multistage tumorigenesis. During the chemically induced differentiation of MEL cells, expression of the c-myb protooncogene is downregulated in a biphasic fashion and forced expression of c-myb is able to block the differentiation process, suggesting that c-myb activity may be limiting for differentiation in MEL cells. We have recently produced stable transfectants in the C19 MEL cell line that carry a dominant interfering myb allele (MEnT) under the control of an inducible mouse metallothionein I (MTH) promoter. Upon inducing expression of MEnT, transfected cells enter a differentiation program and begin to produce alpha-globin mRNA, assemble hemoglobin, and stop proliferating. Differential display was used to compare mRNA expression between parental C19 MEL cells induced to differentiate with hexamethylene bisacetamide (HMBA) and stable transfectants induced to differentiate via expression of MEnT to identify potential Myb target promoters. We identified six candidate cDNAs in this fashion and present evidence that two of these represent genes that are dependent on c-Myb activity for maximal expression in MEL cells.     

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.     

Oxidative stress is involved in hydroxyurea-induced erythroid differentiation

Hydroxyurea (HU), an inhibitor of DNA synthesis, can also induce haemoglobinization in certain erythroid cell lines. In this study, we report that intracellular peroxides levels were increased in HU-treated murine erythroleukaemia (MEL) cells and that l-acetyl-N-cysteine (LNAC), a potent reducing reagent, had a significant inhibitory effect on the HU-mediated induction of beta-globin, delta-aminolaevulinate synthase mRNA expression and haemoglobinization of MEL cells. In contrast, the addition of LNAC to dimethyl sulphoxide (DMSO)-treated MEL cells had a much smaller effect on the number of haemoglobinized cells. These findings suggest that oxidative stress is involved in HU-mediated induction of erythroid differentiation and that HU induces MEL cell differentiation by a mechanism different to that involved in DMSO-mediated differentiation. Our findings also suggest that the induction of MEL cell differentiation by HU does not involve RAS-MAP (mitogen-activated protein) kinase signalling.     

Induction of a Ca2+, Mg2+-dependent endonuclease activity during the early stages of murine erythroleukemic cell differentiation.

A Ca2+, Mg2+-dependent endonuclease activity was detected in erythroleukemic cells undergoing differentiation in vitro in response to induction by dimethyl sulfoxide (Me2SO) or hexamethylene-bis-acetamide (HMBA). The endonuclease activity was demonstrated in isolated nuclei within 6 hr after the addition of inducer, reached maximum levels between 24 and 48 hr, and returned to control levels within 72 hr. The activity caused single strand breaks in high molecular weight native DNA, which could be labeled at exposed 3'-OH termini with Escherichia coli DNA polymerase I and radiolabeled nucleotides. Alkaline elution studies revealed DNA fragmentation that appeared coincident with the presence of the endonuclease activity. The detection and levels of single strand DNA breakage correlated with induction of terminal differentiation by Me2SO or HMBA. Induction of the endonuclease activity was reversible: depletion of Me2SO from the growth medium after treatment for 6 and 18 hr led to a rapid decrease in the level of activity. Removal of the inducer prevented terminal differentiation, a finding that strongly suggests the endonuclease activity is present during the precommitment phase of differentiation. DNA fragmentation was not observed in cells incubated with hemin, which has been shown previously to increase the cytoplasmic level of globin mRNA without causing commitment to terminal maturation. Me2SO did not induce the endonuclease activity or DNA fragmentation in an uninducible Friend cell line.     

Bb1-3, a transgenic hybrid cell line with erythroid and megakaryocytic differentiation potential that expresses high levels of human γ-globin and human β-globin

We have characterized a murine hybrid cell line, Bb1-3, generated by the fusion of mouse primary erythroblasts with MEL cells. It proliferated in serum-free medium and displayed a low level of spontaneous erythroid and megakaryocyte differentiation. Terminal erythroid differentiation could be induced with HMBA and DMSO and was enhanced by serum. Treatment with phorbol esters resulted in a high proportion of megakaryocytes and the expression of megakaryocytic specific lineage markers. Bb1-3 cells contain a human β-globin transgene that was expressed at levels of 20–50% of the endogenous mouse globin genes. Initially, expression was largely limited to the β-globin gene but after adaptation to serum free growth, equal expression of both the human γ- and human β-globin genes was observed. This cell line provides further evidence that the differentiation potential of mouse erythroleukaemia cells is not restricted to the erythroid lineage and should be useful to study the mechanisms underlying both developmental globin gene regulation and the terminal differentiation of bipotential erythroid/megakaryocytic progenitor cells.     

Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells.

The only known role for 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) is as the cofactor for the aromatic amino acid hydroxylases. However, BH4 has been shown to be synthesized by cells that do not contain any hydroxylase activity, suggesting that it may have still undiscovered functions. Our finding of much higher levels of BH4 and GTP cyclohydrolase, the first enzyme of de novo BH4 biosynthesis, in rat reticulocytes compared to mature erythrocytes raised the possibility that BH4 might play a role in erythrocyte maturation. We have now demonstrated, by using murine erythroleukemia (MEL) cells as a model for erythrogenesis, that BH4 synthesis is required for proliferation of these cells. Inhibition of BH4 biosynthesis in rapidly dividing MEL cells with N-acetylserotonin, a potent inhibitor of sepiapterin reductase, the terminal enzyme in the BH4 biosynthetic pathway, results in inhibition of DNA synthesis and mitogenesis without induction of hemoglobin synthesis. The inhibition of DNA synthesis is reversed by repletion of cellular BH4 levels with sepiapterin, a pterin that is readily taken up by the cells and converted to BH4 by the sequential reductions of sepiapterin reductase and dihydrofolate reductase. Treatment of MEL cells with hexamethylene bisacetamide, an inducer of differentiation, results in a decrease in BH4 synthesis accompanied by a cessation of growth and concomitant hemoglobin synthesis. The inhibition of proliferation induced by hexamethylene bisacetamide can be reversed by maintaining high intracellular levels of BH4, which also decreases the amount of hemoglobin. The mechanism of the BH4 effect has not yet been elucidated, but it appears as though BH4 synthesis is more intimately linked with cell proliferation than with the differentiation process.