Antagonistic effect of butyrate on hexamethylene bisacetamide induced differentiation of murine erythroleukemia cells.

Butyrate, at concentrations greater than 0.75 mM, induces hemoglobin accumulation in murine erythroleukemia cells (MELC). At concentrations below 0.75 mM, butyrate inhibits hemoglobin accumulation induced by hexamethylene bisacetamide (HMBA) as well as HMBA induced commitment to terminal cell division. The blocking of HMBA induced differentiation does not result from growth inhibition. When cells were exposed to HMBA and butyrate for 4 days and then both inducers were removed, the cells did not terminally divide. On the other hand, cells exposed to HMBA for 4 days, with subsequent removal of HMBA, did go on to terminally divide. Thus, butyrate blocks the ability of HMBA to accumulate the intracellular signals for terminal cell division. A 48-h pretreatment of cells with butyrate did not inhibit the ability of subsequent HMBA treatment, after butyrate removal, to induce terminal cell division. These results might suggest that cells do not generate a memory of exposure to HMBA in the presence of butyrate or a memory of exposure to butyrate when used as a pretreatment under these conditions. HMBA, at concentrations below 1.0 mM, does not induce MELC differentiation, but such concentrations actually enhance dimethyl sulfoxide induced differentiation of MELC. Equimolar concentrations of short chain fatty acids (1 to 7 carbons) were tested for their ability to block HMBA induced differentiation of MELC. Butyrate and valerate (4 and 5 carbons, respectively) had blocking activities similar to each other, whereas the other fatty acids exhibited little or no blocking of HMBA induced differentiation.

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells.

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.

Expression of growth hormone-independent adipogenesis by a 3T3 cell variant.

We have examined the regulation of adipogenesis of a 3T3-F442A cell variant. The variant, designated 3T3-GH-independent clone 16 (GI-16), was isolated after serum-induced adipogenic commitment. 3T3-GI-16 fibroblasts displayed a lower serum requirement for adipogenesis than the 3T3-F442A parent cell. Insulin-stimulated adipogenesis of 3T3-GI-16 cells in serum-free medium (SFM) was extensive in the absence of GH, as judged by oil red O staining or glycerol-3-phosphate-dehydrogenase activity, a property not associated with the 3T3-F442A cell. In SFM devoid of GH the concentration of insulin required to promote half-maximal adipogenesis of 3T3-GI-16 fibroblasts was 5 nM. The expression of GH-independent adipogenesis by 3T3-GI-16 cells was not due to exposure to adipogenic stimuli during routine passage, as insulin-stimulated differentiation was not a function of the inoculation density in nonadipogenic cat serum. We noted that nine proteins resolved by polyacrylamide gel electrophoresis behaved in a differentiation-dependent manner during adipogenesis of 3T3-GI-16 and 3T3-F442A fibroblasts in SFM. The concentrations of all nine proteins were regulated in a GH-independent manner during insulin-stimulated adipogenesis of 3T3-GI-16 fibroblasts. In contrast, the presence of insulin alone markedly altered the expression of only two of the proteins during differentiation of 3T3-F442A cells. The observed changes in the expression of five presently uncharacterized differentiation-dependent proteins were most likely due to employment of SFM. Our results suggest that expression of GH-independent insulin-induced adipogenesis of 3T3-GI-16 fibroblasts reflects a prior commitment by GH during our selection protocol. These results are discussed in the context of a model in which adipogenesis in vivo is postulated to proceed through the sequential action of GH and insulin on target cells.

Up-regulation of vinculin expression in 3T3 preadipose cells by growth hormone.

In an effort to define biochemical events relevant to the adipogenic action of GH, the effect of GH on expression of the cytoskeletal element vinculin was studied in 3T3-F442A preadipose cells. Results from Western blotting indicated that in serum-free medium 2 nM met-hGH induced an approximately 100% increase in vinculin expression relative to that in cells maintained in serum-free medium alone. GH-elicited alterations in vinculin expression were dose dependent. GH treatment elevated levels of tubulin to a lesser extent, whereas actin expression was unaffected by GH. Immunoprecipitation experiments revealed that GH treatment promoted a 200% increase in vinculin synthesis on day 4 relative to that in control cells. GH had no effect on phosphorylation of vinculin in 3T3-F442A cells. Based on Northern blotting, we noted that GH induced approximately a 200% increase in levels of vinculin mRNA on day 4. GH responsiveness as well as levels of vinculin were similar in 3T3-GI-16 (a highly adipogenic subclone of the 3T3-F442A cell) and 3T3-F442A cells. The GH-dependent increase in vinculin protein expression was not observed in nonadipogenic 3T3-C2 cells, suggesting that this effect of GH was related to the program of differentiation. Interestingly, levels of vinculin in nontreated 3T3-C2 cells were approximately 10-fold lower than levels in 3T3-F442A cells. GH-mediated alterations in vinculin expression in 3T3-F442A cells were abolished by treatment with fetal bovine serum, a potent mitogen. Our data indicate that increased expression of vinculin is a component of the GH-induced portion of the adipose differentiation program.

Antagonism by growth hormone of insulin-sensitive hexose transport in 3T3-F442A adipocytes.

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.

Role of insulin in growth hormone-stimulated 3T3 cell adipogenesis.

The role of insulin during GH-stimulated adipogenesis of 3T3-F442A fibroblasts was investigated. Adipogenesis in defined medium (DM), as quantified by the level of glycerol-3-phosphate dehydrogenase activity, revealed that there existed a strict requirement for both insulin and GH during adipogenesis. The concentration of insulin required to elicit half-maximal adipogenesis was approximately 20 nM. Insulin-like growth factor I was less effective than insulin in promoting adipogenesis, indicating that insulin action during differentiation was most likely mediated through the insulin receptor. Cellular viability was not compromised by the absence of insulin, as judged by colony-forming efficiency or trypan blue exclusion. Deletion of insulin from DM supplemented with 1 nM recombinant human GH reduced glycerol-3-phosphate dehydrogenase activity to uninduced levels. Removal of other individual DM constituents did not have this effect. The growth factors fibroblast growth factor, platelet-derived growth factor, and bombesin did not substitute for insulin during GH-stimulated adipogenesis. The characteristic increase in cell number observed during serum-based differentiation, reflecting clonal expansion of young adipocytes, did not occur in DM supplemented with insulin, and insulin-like growth factor I were necessary for this event. These results suggest that insulin functions in concert with GH as a coinducer of the differentiating signals.

Growth hormone-dependent events in the adipose differentiation of 3T3-F442A fibroblasts: modulation of macromolecular synthesis.

GH is necessary but not sufficient to induce adipose differentiation of 3T3-F442A fibroblasts in serum-free medium. Human (h) GH (2 nM) treatment of 3T3-F442A cells in serum-free medium caused a time-dependent (maximal at 48-72 h) and dose-dependent (EC50, approximately 0.2 nM) decrease (40-60%) in de nova protein synthesis. Insulin-like growth factor-I (IGF-I; 17 nM), PRL (2 nM), and glucagon (20 nM) did not decrease de novo protein synthesis, whereas bovine GH was equipotent with hGH. The half-lives of 35S-labeled proteins of 3T3-F442A cells were 21 and 57 h for cells maintained in serum-free medium for 3 days without or with hGH (2 nM), respectively. The total protein content of cells maintained in hGH (2 nM) for 1-4 days was unaffected compared to cells in serum-free medium alone. IGF-I (17 nM) treatment of cells for 4 days doubled the protein content of cells compared to control values in serum-free medium. hGH (2 nM) pretreatment of cells for 1-4 days had no effect on total RNA synthesis. hGH (2 nM) but not IGF-I (17 nM) treatment (3 days) resulted in a 7-fold decrease in cytoplasmic 18S rRNA content (as measured by DNA-RNA hybridization) of cells compared to that of control cells maintained in serum-free medium. When 3T3-F442A cells were transferred to serum-free medium there was a progressive decrease in DNA synthesis. The presence of hGH enhanced the rate at which DNA synthesis decreased for 3T3-F442A cells. IGF-I (17 nM) increased DNA synthesis by 6- and 8-fold after 2 and 3 days of IGF-I exposure. 3T3-F442A cells maintained in serum-free medium for 3 days responded to the addition of platelet-derived growth factor (2 U/ml) and insulin (1.6 microM) with a 56-fold increase in DNA synthesis, assayed 24 h later. 3T3-F442A cells treated with hGH (2 nM) for 3 days before platelet-derived growth factor and insulin addition exhibited a diminished DNA synthetic response, demonstrating that GH-exposed cells were partially refractory to mitogenic stimulation. GH had no effect on any aspect of macromolecular synthesis in 3T3-C2 cells, which have a low frequency of adipogenesis. Based upon these results a cell cycle model for the role of GH in the adipose differentiation of 3T3-F442A cells was proposed.

Growth hormone and fibronectin expression in 3T3 preadipose cells.

In the present study we focused on the relationship between GH action and the extracellular matrix in 3T3-F442A preadipose cells. Results from Northern blotting indicated that in serum-free medium, the presence of 2 nM met-human GH down-regulated levels of fibronectin messenger RNA by approximately 40, 60, and 70% as compared with control levels on days 1, 2, and 4, respectively. GH-dependent reduction of levels of collagen alpha 1(I) mRNA expression occurred later and was less pronounced than effects on levels of fibronectin mRNA, suggesting a specificity in the matrix-altering function of GH. Western blot analyses and immunoprecipitation studies revealed that between 2 and 5 days of culture, matrix-associated fibronectin protein was reduced 70 to 90% by GH treatment. Down-regulation of fibronectin protein expression by met-human GH was dose-dependent between 2 and 0.02 nM. The presence of 2 nM insulin or insulin-like growth factor-1 promoted a 30-40% increase in fibronectin levels compared to control cells. The GH-promoted down-regulation of fibronectin expression was eliminated by concomitant addition of insulin. These data demonstrated that GH effects on matrix-associated fibronectin expression were independent of, and in opposition to, effects promoted by insulin and insulin-like growth factor-1. Treatment of culture dishes with fibronectin or collagen inhibited GH-stimulated adipogenesis 50 and 80%, respectively, compared with controls, as judged by levels of glycerol-3-phosphate dehydrogenase activity. Thus, composition of the extracellular matrix was a critical factor in GH-induced adipogenesis of 3T3-F442A fibroblasts. Our results demonstrate that GH action in 3T3 preadipose cells is intimately coupled to the biology of extracellular matrix.

Activity of growth hormone peptides bGH 96-133 and hGH 95-133 in 3T3-F442A cells.

Chemically synthesized bovine growth hormone (bGH) bGH 96-133 and its human homologue, hGH 95-133, have similar in vitro biological activities. Unlike native GH, bGH 96-133 and hGH 95-133 were completely without adipogenic or anti-insulin activity at doses up to 10 microM. bGH 96-133 had insulin-like activity, with a 100% increase in glucose uptake at 10 microM. bGH was anti-mitogenic and bGH 96-133 and hGH 95-133 were mitogenic (EC50 approximately 180 nM and maximal response at 1-2 microM). Only bGH 96-133 and hGH 95-133 displaced [125I]hGH 95-133 binding from 3T3-F442A fibroblasts with a Kd between 60-120 nM. bGH, hGH, insulin and IGF-I were without effect on [125I]hGH 95-133 binding. bGH 96-133 and hGH 95-133 did not significantly inhibit [125I]hGH or [125I]IGF-I binding. These experiments indicate that GH containing peptides bGH 96-133 and hGH 95-133 have mitogenic and insulin-like activity without the adipogenic, anti-insulin or anti-mitogenic activity of bGH. These peptides have a specific binding site which appears to be distinct from the GH, insulin and IGF-I receptors.

Anti-fertility and other actions of gossypol analogues.

From a series of gossypol derivatives studied, we conclude that the carbonyl groups of gossypol are needed for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were bi-aromatic, linked by a 1- and not a 4-carbon chain and had free phenolic hydroxyl groups. These results are included in a discussion of the specificity and mechanism of action of gossypol.

PIP: The analysis of multiple biological assays of gossypol and its derivatives suggests that the carbonyl groups of gossypol are required for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were biaromatic, linked by a 1- and not a 4-carbon chain, and had free phenolic hydroxyl groups. When gossypol inhibits the anion transporter, the carbonyl group does not seem to form a Schiff base. Gossypol is a unique compound since it alone, but not any of its derivatives, has in vivo as well as in vitro antifertility activity. It remains unknown, however, whether similar mechanisms are involved in gossypol's in vivo and in vitro effects. In whatever manner gossypol exerts its toxic effects, the selectivity for testicular tissue must be explained.

Characterization of leptospiral catalase and peroxidase.

Peroxidase from Leptospira biflexa strain B-16 ad catalase from Leptospira interrogans canicola Hond Utrecht were characterized and compared and both appeared to be heme enzymes as judged by their inhibition profiles and rapid inactivation during catalysis. Neither enzyme exhibited monovalent or divalent cation requirements. Dialysis of cell-free extracts resulted in loss of peroxidase activity but catalase was unaffected by this procedure. Peroxidase had a Km for H2O2 of 12.5 microM while catalase had a Km of 70 mM for H2O2. Catalase and peroxidase were physically separated by sedimentation in linear sucrose gradients. The specific activities of each enzyme seemed to be a function of the state of growth at which the cells were harvested and both enzymes were found associated with membranes, peroxidase by hydrophobic and catalase by ionic interactions. Speculative deductions are presented concerning the phylogenetic interrelationships of both enzymes as well as their significance in the biology and pathogenicity of leptospires.

Formation of a receptor state from which insulin dissociates slowly in hepatic cells and plasma membranes.

125I-insulin dissociated from rat hepatocytes and liver plasma membranes with a time course suggestive of more than a single kinetic process. Dissociation curves were resolved into rapidly and slowly dissociating components. Increasing times of hormone-cell or hormone-membrane incubation prior to the initiation of dissociation increased the proportion of slowly dissociable 125I-insulin and decreased the proportion of rapidly dissociating hormone. The rates of loss of rapidly and slowly dissociating 125I-insulin, 1 to 2 x 10(-3) and 2 to 7 x 10(-5) s-1, respectively, were the same in cell and membrane incubates. The capacity of liver membranes and hepatocytes to bind 125I-insulin in a slowly dissociable state was saturable with respect to insulin concentration (approximately 10(-8) M). The observation of the same physical process in both cells and plasma membranes demonstrates a distinct role for receptors at the exterior surface of target cells in the retention of insulin.

Up-regulation of insulin receptors in rat liver plasma membranes.

Kinetic experiments (uptake versus time) were utilized to examine the effects of occupancy on insulin receptor availability in rat liver plasma membranes in vitro. The following observations were made: 1) at 4 degrees C, a 3-h exposure of membranes to 100 nM native insulin, followed by removal of unbound hormone, resulted in a subsequent decrease of 125I-insulin binding at 4 degrees C. In a similar experiment at 23 degrees C, no decrease of 125I-insulin binding was observed. 2) At 23 degrees C, 131I-insulin (5 nM) was bound to membranes in a slowly reversible manner after a 3-h association. After removal of free hormone, the 131I-insulin-treated membranes displayed similar binding of 125I-insulin (1 nM) relative to controls despite persistent high level occupancy of receptors by 131I-insulin. 3) At 23 degrees C, phospholipase pretreatment of membranes enhanced 125I-insulin uptake (approximately 40%). Phospholipase-digested membranes exposed to 100 nM native insulin for 3 h bound more 125I-insulin (approximately 40%) than did nondigested membranes preincubated without native insulin. The results allowed speculation that rat liver membranes up-regulated insulin receptors after treatment with insulin and that this was mediated by exposure of cryptic binding sites.

Insulin binding to liver plasma membranes from rats rendered diabetic by alloxan. A kinetic demonstration of two classes of binding sites in equilibrium with each other.

The association of 125I-labelled insulin with liver plasma membranes from diabetic rats was consistent with more than one compartment of binding. After a short association period, insulin dissociation comprised rapid and slow phases. After a long association period, dissociation was only at a slow rate. Lower-affinity hormone-receptor complexes were converted to higher-affinity complexes as the time of occupancy lengthened.

Insulin receptors convert to a higher affinity state subsequent to hormone binding. A two-state model for the insulin receptor.

Kinetic experiments were performed to determine the effects of insulin receptor occupancy on insulin binding. The following results were obtained: (a) the rate constant (k1) for uptake of 125I-insulin by liver plasma membranes was 2 X 10(6) M-1 s-1 and invariant at applied hormone concentrations of 7.5 to 100 X 10(-11) M. 125I-Insulin dissociated from membranes in a biphasic manner with rapid (k-1 = 2-4 X 10(-3) s-1) and slow (k-1 = 2-3 X 10(-4) s-1) components of release when dissociation was initiated by dilution into excess medium. Under all dissociation conditions employed, 125I-insulin was the radioactive species bound to and released from membranes. (b) Native insulin (100 nM) or 131I-insulin (5 nM) in the dissociation medium enhanced the dilution-induced dissociation of bound 125I-insulin. In the latter experiment, total receptor occupancy (bound 125I-insulin and 131I-insulin) decreased during dissociation. The enhanced dissociation effect was therefore not necessarily due to increased site occupancy. (c) As association time prior to dissociation was increased, the dissociability of bound 125I-insulin diminished. Decreased dissociability resulted from an increase in the slow component of hormone release at the expense of the rapid component. 131I-insulin was bound to membranes to which 125I-insulin had been prebound. The dissociation of 131I-insulin had been prebound. The dissociation of 131I-insulin was unaffected by the presence of 125I-insulin dissociating at either rapid or slow rates. The data suggest that there are no cooperative interactions between binding sites and that the hormone-receptor complex converts to a higher affinity state subsequent to occupancy since KD = k-1/k1 and K-1 decreased. A two-state model for the hepatic insulin receptor is proposed.

Enzymatic degradation of H2O2 by Leptospira.

The enzymes responsible for reducing H2O2 were surveyed in 49 strains of Leptospira by using semiquantitative assays for catalase and peroxidase. The survey revealed a differential distribution of catalase and peroxidase activities between the two leptospiral complexes. The pathogenic Leptospira interrogans strains gave strong catalase and weak or negative peroxidase reactions. Conversely, the nonpathogenic Leptospira biflexa strains gave strong peroxidase and negative or weak catalase reactions. An intermediate group of four L. biflexa strains, which were isolated from mammals, fell into the high peroxidase, low or negative catalase group. One water isolate, H-23, gave strong reactions for both enzymes and was examined for virulence and in vitro growth parameters. Results indicate metabolic differences between pathogens and water forms in their abilities to reduce H2O2.

Effect of gossypol on erythrocyte membrane function: specific inhibition of inorganic anion exchange and interaction with band 3.

The effects of gossypol on membrane functions of the human erythrocyte were studied. Gossypol (10 microM) had no effect on spontaneous hemolysis, osmotic fragility, cell volume, cholinesterase activity, hexose transport, ouabain-sensitive inorganic cation transport, ouabain-insensitive inorganic cation transport and nucleoside transport. Conversely, 10 microM gossypol inhibited inorganic anion transport by approximately 90% for three different substrates, i.e., phosphate, sulfate and chloride. Inhibition of inorganic anion transport was specific as 10 microM gossypol had no effect on the eight aforementioned membrane-related functions of the human erythrocyte. Inhibition inorganic anion transport was characterized using sulfate as the substrate and had the following features: it was potent, with a Ki of approximately 3 microM; it was rapid, with onset occurring in less than 1 min; it was potently blocked by physiological concentrations of albumin and plasma with 50% blocking achieved at 0.03% (w/v) albumin; it occurred by a noncompetitive kinetic mechanism; it was independent of medium Ca++, Mg++ or pH. Gossypol was bound to human erythrocytes and cell membranes isolated from erythrocytes. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid is a potent inhibitor of anion transport and can be covalently bound to band 3. Covalently bound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid blocked a fraction of gossypol binding to erythrocyte membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate.

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of the folate compound methotrexate decreases during differentiation of murine erythroleukemia cells.

The relationship of folate transport and chemically induced differentiation of murine erythroleukemia cells (MELC) was examined. MELC were found to have a carrier-mediated transport system for reduced folates. Chemically induced differentiation of MELC caused a 10-fold decrease in the rate of influx of the folate analog methotrexate. The loss of methotrexate transport during differentiation resulted from a 15-fold decrease in the Vmax for the influx while the Km remained unchanged. Inducer dose dependencies for hemoglobin accumulation and loss of methotrexate influx were similar. A dimethyl sulfoxide-resistant variant of MELC did not express complete accumulation of hemoglobin or loss of methotrexate transport when treated with dimethyl sulfoxide. The decrease of folate influx during differentiation was not a result of: 1) changes in cell densities or growth phase; 2) growth-related changes of the medium; or 3) a direct inhibition of folate influx by the inducers. Loss of folate compound transport correlated with MELC differentiation.

Cytotoxicity of crotoxin on murine erythroleukemia cells in vitro.

The cytotoxic effect of crotoxin, a heterodimeric phospholipase A2 from the venom of Crotalus durissus terrificus, was examined on murine erythroleukemia cells in vitro. Crotoxin cytocidal effect on cell growth had an EC50 of approximately 0.1-0.2 microM (3.0-5.0 micrograms/ml) in serum-free medium. Cytotoxicity was independent of cell growth since both quiescent and proliferating cells had similar sensitivities to the toxin. Dissociation of the crotoxin complex and phospholipase A2 activity of its subunit B are required for cytotoxicity, since the covalently linked crotoxin complex or the specific alkylation of the active site on the subunit B abolish the cytotoxic activity on murine erythroleukemia cells. Specific interaction between crotoxin and murine erythroleukemia cells appears to be required since the homologous phospholipase A2 from Crotalus atrox venom, with a higher phospholipase A2 specific activity than crotoxin, was 86-fold less potent than crotoxin. The data in this report show that the cytotoxic effect of crotoxin on murine erythroleukemia cells is consistent with the specific binding of the toxin resulting in cytocidal action mediated by the phospholipase A2 activity of crotoxin subunit B.