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.     

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.     

Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester.

Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylates serine residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 micrograms of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal insulin-stimulated S6 kinase has a Mr of approximately equal to 50,000-60,000; there is a variable amount of activity that sediments with a Mr of about 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2+ (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.05 mM), and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 micrograms/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate 13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-L1 cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of Mr 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. The data suggest that the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid, and Ca2+-independent proteolytic derivative nor the result of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.     

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.     

Relationship between transient DNA hypomethylation and erythroid differentiation of murine erythroleukemia cells.

The state of DNA methylation in mouse erythroleukemia (MEL) cells has been analyzed in relation to commitment to differentiation in response to treatment with hexamethylenebisacetamide (HMBA). Previous experiments have shown that induction by HMBA involves transient genome-wide hypomethylation of DNA that is achieved by replacement of 5-methylcytosine with cytosine residues. The experiments described in the present communication revealed that hypomethylation is a very early event in the process of differentiation. Exposure of the cells to 3-deazaadenosine, an adenosine analog, in combination with homocysteine, resulted in the intracellular accumulation of 3-deazaadenosylhomocysteine, which caused an inhibition of HMBA-induced hypomethylation that was correlated with a comparable inhibition of differentiation. While these experiments suggest that hypomethylation is a necessary step in the process of differentiation, other experiments reported here indicate that hypomethylation of DNA may be necessary but not sufficient to trigger the whole program of differentiation in MEL cells. We found, for example that exposure of the cells to cycloheximide during the first 24 hr of induction by HMBA resulted in complete inhibition of differentiation without significant effect on the HMBA-induced hypomethylation. This result also indicates that the enzymatic machinery required for the hypomethylation of DNA is present in uninduced cells.     

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.     

Activation of Na+/H+ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca2+ ionophore, and growth factors.

The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H+ exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H+ exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts.     

Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase.

In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2+ and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction.     

Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control.     

Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin. PMA also enhanced secretion induced by Ba2+. In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+. PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30%. The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.     

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.     

Dexniguldipine hydrochloride,a protein-kinase-C-specific inhibitor,affects the cell cycle,differentiation, P-glycoprotein levels,and nuclear protein phosphorylation in friend erythroleukemia cells

Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance. Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity. Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation. P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance. At 2.5 M, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase. DNA synthesis was decreased in cells exposed to DNIG for 20 h. Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle. Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug. HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation. Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein. western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC. Finally, levels of P-glycoprotein were decreased in the presence of this drug. Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation. Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance. The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.Abbreviations DNIG dexniguldipine hydrochloride - FELC Friend erythroleukemia cells - HMBA hexamethylene bisacetamide - MDR multidrug resistance - PKC protein kinase C Financial support: this work was supported in part by a grant from the Ladies Leukemia League to J. R. J. and a grant from the NIH, DK40501, to B. S. B.     

Demonstration of protein kinase C activity in crustacean Y-organs, and partial definition of its role in regulation of ecdysteroidogenesis

Ecdysteroid-producing Y-organs from the crab Cancer antennarius were shown to possess enzyme activity that was stimulated in vitro by addition of Ca2+, phosphatidylserine, or the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; ED50, 4 nM). In the presence of calcium and phosphatidylserine, PMA increased protein kinase C activity dose-dependently to a maximum 4-fold increase at 100 nM PMA. Stimulated protein kinase C activity was unaffected by calmodulin (100 nM) but was inhibited by 100 nM trifluoperazine. Pretreatment of cultured Y-organ segments with PMA elevated basal protein kinase C activity, whereas molt-inhibiting hormone (MIH) and calcium ionophore A23187 did not affect activity. PMA (1-100 nM) increased Y-organ steroidogenesis dose-dependently and alleviated suppression due to MIH or lysine vasopressin; PMA effects on steroidogenesis became evident after 2 h of incubation. Another phorbol activator of protein kinase C (phorbol 12, 13-dibutyrate) and a permeable synthetic diacylglycerol (1-oleoyl-2-acetyl-glycerol) stimulated ecdysteroidogenesis while an inactive phorbol (4 alpha-phorbol 12,13-didecanoate) and diolein were ineffective. The inhibitory effects on steroidogenesis of cholera toxin, forskolin, dibutyryl cAMP, and 3-isobutyl-1-methylxanthine were countered by PMA, but PMA did not alter basal or peptide hormone-stimulated Y-organ cAMP levels. Stimulatory effects on steroidogenesis of PMA and of A23187 were not additive, and PMA did not alter inhibition caused by lanthanum (calcium channel blocker) or trifluoperazine (calmodulin inhibitor). PMA increased the incorporation of [3H]leucine into Y-organ protein by 112%, and countered the suppressive effect of MIH on protein synthesis; PMA did not affect RNA synthesis. When Y-organs were suppressed with cycloheximide, PMA was unable to stimulate steroidogenesis. Actinomycin D alone had no effect on steroidogenesis but prevented stimulation by PMA. The results indicate that Y-organs contain protein kinase C activity which stimulates ecdysteroid production and protein synthesis by a mechanism not directly interactive with the cAMP or Ca2+-calmodulin systems.     

Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2(+)-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10(-6) M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 +/- 11 nM in PMA-treated cells compared to 703 +/- 44 nM in control cells. High extracellular Ca2(+)-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10(-6) M) increased [Ca2+]i to an initial peak of 738 +/- 49 nM followed by a sustained increase to 501 +/- 30 nM in control cells (n = 15). After exposure to PMA (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 +/- 5 and peaked at 323 +/- 15 nM with ionomycin (10(-6) M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 +/- 19 nM observed with ionomycin (10(-6) M) in control cells (P less than 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2(+)-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.

Incubation of the cytoskeletal fraction from human neutrophils with the proteolytically activated form of protein kinase C results in the phosphorylation of several components, including a 20-kDa polypeptide, probably consisting of myosin light chains. The 20-kDa polypeptide is also specifically phosphorylated by activated protein kinase C in a solubilized 20-kDa/80-kDa complex that was obtained after sonication of the insoluble cytoskeletal fraction. Phosphorylation of this polypeptide, in either the insoluble cytoskeletal fraction or the soluble 20-kDa/80-kDa complex, greatly enhances its susceptibility to digestion by the Ca2+-requiring proteinase (calpain, EC 3.4.22.17) of human neutrophils. Thus, signals that activate calpain by mobilizing intracellular calcium would lead to proteolytic activation of protein kinase C, phosphorylation of cytoskeletal proteins, and remodeling of the cytoskeleton by proteolysis of at least one cytoskeletal component.     

Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.     

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.