Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells.

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.

Cyclopentenyl cytidine analogue. An inhibitor of cytidine triphosphate synthesis in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of cytidine, cCyd, was investigated in human colon carcinoma cell line HT-29. Upon exposure of cells to 10(-6)M cCyd, cell viability was reduced to 20% of control, whereas cytocidal activity was not present after 2 hr of drug exposure. Cell lethality was partially reversible by Urd, Cyd or dCyd at 10(-6)M cCyd, and fully reversible by these nucleosides at 2.5 X 10(-7)M cCyd. The incorporation of [14C]dThd and [3H]Urd into DNA and RNA was inhibited by 50% by exposure for 2 hr to 2.5 X 10(-7) and 1.5 X 10(-6)M cCyd respectively. Upon 24 hr of drug exposure, the IC50 for RNA synthesis was reduced 2.5-fold, whereas DNA synthesis was almost totally inhibited. cCyd produced a rapid and preferential reduction of CTP synthesis with a half-life of 1 hr at 10(-6)M drug. The IC50 of cCyd for reducing CTP concentrations after 2 hr of drug exposure was 4 X 10(-7)M. Concomitant with the reduction of CTP levels was the inhibition of transcription of rRNA and, to a lesser extent, tRNA, without changes in the processing nucleolar RNA. No changes in the size of DNA were produced following treatment with cCyd. These results indicate that cCyd is a potent and rapid inhibitor of CTP synthesis and that this effect correlates with its cytocidal activity.

3-Deazaneplanocin A: a new inhibitor of S-adenosylhomocysteine synthesis and its effects in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of 3-deazaadenosine (3-deazaneplanocin A or c3Nep) was investigated in the human colon carcinoma cell line HT-29. Upon exposure of cells for 24 hr to 3-deazaneplanocin A (c3Nep), neplanocin A (Nep) or 3-deazaaristeromycin (c3Ari), significant toxicity was noted only for Nep, wherein an 87% reduction in viability was produced at a 100 microM concentration. c3Nep and c3Ari at 100 microM reduced viability by 34 and 21%, respectively. Intracellular levels of S-adenosylhomocysteine (AdoHcy) were elevated by a 24-hr exposure to 100 microM c3Nep, Nep and c3Ari and were 120, 75 and 25 pmoles/10(6) cells respectively. Only Nep was metabolized to an S-adenosylmethionine-like metabolite, and its formation was dose-related to its cytotoxicity. The t1/2 for the disappearance of elevated levels of AdoHcy following drug removal was 1.6 to 2.5 hr for all drugs. rRNA and tRNA methylation was inhibited significantly by Nep, but c3Nep and c3Ari inhibited tRNA methylation but not rRNA methylation to a lesser degree. These results demonstrate that c3Nep is a potent inhibitor of AdoHcy synthesis with a low degree of cytotoxicity.

Induction of differentiation in the human promyelocytic leukemia cell line HL-60 by the cyclopentenyl analogue of cytidine.

The effects of the cyclopentenyl (cCyd) and cyclopentyl (carbodine) analogues of cytidine on differentiation, and nucleic acid and nucleotide biosynthesis, were examined in the human promyelocytic leukemia cell line HL-60. Continuous exposure for 5 days to 10(-8) to 10(-6) M cCyd or 10(-6) to 10(-5) M carbodine produced progressive inhibition of cell growth. During this exposure interval, pronounced differentiation to mature myeloid cells occurred wherein 95% of the cell population reduced nitroblue tetrazolium 4 days after exposure to 10(-7) M cCyd or 10(-5) M carbodine. Preceding differentiation was the inhibition of DNA synthesis which reached 10% of control levels 24 hr after exposure to 10(-7) M cCyd or 10(-5) M carbodine, while RNA synthesis was inhibited to a lesser extent. The induction of mature myeloid cells by cCyd was preceded by the inhibition of c-myc mRNA levels which was more pronounced than the reduction in total cellular RNA synthesis. During the interval of cCyd treatment, there was a rapid and pronounced inhibition in the level of CTP, but not of UTP, ATP or GTP, where the half-life for the disappearance of CTP was 1.5 to 2 hr. Following drug removal, cells treated with cCyd showed a sustained reduction in CTP levels, whereas cells treated with carbodine showed almost complete recovery of CTP levels within 48 hr. These results indicate that the reduction in CTP levels leads to rapid inhibition of DNA synthesis and reduction in c-myc mRNA levels which precede the appearance of differentiated cells.

Retinoic acid promotes phorbol ester-initiated macrophage differentiation in HL-60 leukemia cells without disappearance of protein kinase C.

The ability of retinoic acid (RA) to promote 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-initiated macrophage differentiation was examined in human promyelocytic leukemia cell line HL-60. One-hour exposure to 10 nM TPA and subsequent exposure for 48 h to 1 microM RA following removal of TPA rapidly induced the macrophage phenotype in 65% of the cells. This effect was comparable to continuous exposure for 48 h to TPA alone, but contrasted with the absence of macrophage-like cells after RA treatment alone or the induction of 10% of the cell population to a macrophage phenotype after 1-h exposure to TPA. The effect of TPA + RA was accompanied by increased cell adherence and increased nonspecific esterase activity but not by a change in the reduction of nitroblue tetrazolium. Protein kinase C (PK-C) activity was increased 35-40% in cells treated for 1 h with TPA alone or after subsequent exposure to RA. Cells treated for 48 h with RA exhibited a 2-fold increase in PK-C activity while cells exposed to TPA for 48 h lost all PK-C activity. The changes in PK-C activity in TPA + RA-treated cells were accompanied by Ca2+/phospholipid(PL)-dependent phosphorylation in vitro of pp38 which is characteristic of treatment with RA alone, as well as the Ca2+/PL-independent phosphorylation in vitro of pp82 and pp130 (vinculin) which is prevalent in cells treated continuously with TPA alone and is absent in RA-treated cells. These results indicate that the macrophage phenotype induced by TPA + RA is similar to that produced by continuous exposure to TPA alone with respect to their in vitro phosphoprotein patterns, cytochemical markers, cell adherence and morphology, but that the disappearance of PK-C is not an obligatory characteristic of these cells.

1-beta-D-arabinosyl-5-azacytosine. Cytocidal activity and effects on the synthesis and methylation of DNA in human colon carcinoma cells.

The cytocidal activity of arabinosyl-5-azacytosine (araAC) and its effect on the synthesis and methylation of DNA in the human colon carcinoma cell line HT-29 was examined and compared with three other cytidine analogues. Treatment for 2 hr with 10(-6)M arabinosylcytosine (araC), araAC, 5-azacytidine (AZC), or 2'-deoxy-5-azacytidine (dAZC) produced a 7-30% reduction in cell viability. Prolongation of drug exposure to 24 hr significantly enhanced the cytotoxicity of all analogues, and particularly dAZC. AZC and dAZC were potent inhibitors of DNA methylation in the absence of inhibition of DNA synthesis, whereas araC and araAC primarily affected DNA synthesis. RNA synthesis was not affected by any of the analogues. dAZC and AZC were incorporated into DNA to a greater extent than were araC or araAC upon short- and long-term drug exposure, whereas only AZC was incorporated into RNA. These data indicate that araAC appears to behave more as an analogue of araC rather than of dAZC or AZC, wherein it produces rapid inhibition of DNA synthesis and is incorporated into DNA.

Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation.

The mechanism of action of the adenosine analog, neplanocin A (NPC), was investigated in human colon carcinoma cell line HT-29. Cell viability was reduced to 38 and 17% of control by 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. Cytocidal activity was not affected by inhibition of adenosine deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability was the reduced incorporation of [14C]dThd and [3H]Leu, and to a lesser extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA during drug treatment for 24 h with [methyl-3H]Met and [14C]Urd revealed that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA synthesis. RNase T2 digests of total RNA indicated that base and 2'-O-methylation were inhibited to approximately the same degree. Metabolites of NPC were measured by reverse-phase high-performance liquid chromatography and it was found that the major drug metabolite was the drug analog of S-adenosylmethionine with little formation of the respective, S-adenosylhomocysteine metabolite. NPC was utilized to a very small degree for RNA synthesis where only 2 and 30 pmol of NPC/A260 were incorporated into rRNA and tRNA after 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. These results indicate that NPC is metabolized to a metabolite of S-adenosylmethionine which is a poor methyl donor for RNA methyltransferases, and that the accompanying decrease in RNA methylation and protein synthesis appears to be related to its cytocidal activity.

The epidermal growth factor- and interferon-independent effects of double-stranded RNA in A431 cells.

The human epidermoid carcinoma cell line A431, containing an amplification in the epidermal growth factor (EGF) receptor gene, was examined for its sensitivity to the growth inhibitory effects of synthetic double-stranded RNAs (dsRNAs). Poly(I).poly(C), poly(A).poly(U) and rln.r(C13,U)n at 5 to 100 micrograms/ml produced 20 to 60% growth inhibition, whereas poly(ICLC) produced 40 to 80% growth inhibition at 0.05 to 25 micrograms/ml.Poly(I).poly(C) did not cause the secretion of interferon (IFN) into the medium, and addition of polyclonal antibodies to IFN-alpha and IFN-beta did not block the growth inhibition produced by poly(I).poly(C). Clone 29, which proliferates in response to EGF, and clone 29R, which is sensitive to the growth inhibitory effects of EGF, showed sensitivities to the antiproliferative effects of poly(I).poly(C) similar to those of the parent cell line. Incubation of cell membrane extracts with poly(I).poly(C) or treatment of cells with the dsRNA did not affect EGF receptor tyrosine kinase activity. On the other hand, poly(I).poly(C) produced a dose-dependent induction of (2',5')oligo(A) synthetase activity and degradation of 45S preribosomal RNA and 28S and 18S rRNA. These results indicate that the growth inhibitory properties of poly(I).poly(C) in A431 cells are independent of the action of IFN but are associated with degradation of rRNA, an effect that may be related to the (2',5')oligo(A)-RNase L pathway.

9-Deazaadenosine. Cytocidal activity and effects on nucleic acids and protein synthesis in human colon carcinoma cells in culture.

The effect of 9-deazaadenosine (c9Ado) on cell lethality and the synthesis of nucleic acids was investigated in human colon carcinoma cell line HT-29. c9Ado produced a rapid threshold-exponential reduction in colony formation as measured by a soft agar clonogenic assay. This effect was evident after either a 2- or 24-hr exposure interval, and was produced over a very narrow concentration range of drug. Following 2 hr of drug exposure at concentrations producing a 1- to 3-log reduction in cell viability, DNA and RNA syntheses were inhibited 20% and protein synthesis was inhibited 35-50%. The latter effect became quite pronounced in comparison to nucleic acid synthesis 4 hr after drug treatment. Long treatment intervals (24 hr) with concentrations of c9Ado producing similar effects on cell viability resulted in 15-35% inhibition of RNA synthesis, 80-85% inhibition of DNA synthesis, and 60-70% inhibition of protein synthesis. None of these metabolic effects could be accounted for by changes in ribonucleoside triphosphate levels despite the considerable formation of c9ATP. Measurements of the incorporation of [3H] c9Ado into total cellular nucleic acids indicated that the labeling of RNA was 40-80% greater than that of DNA. Polysomal poly(A)RNA contained 300% more [3H]c9Ado than non-poly(A)RNA after 2 hr of drug exposure and 50% more [3H]c9Ado following 24 hr of treatment. There was no evidence of DNA strand breakage by incorporated c9Ado. Analysis of nascent protein synthesis in drug-treated cells revealed that this process was inhibited in concert with polysome breakdown. These results suggest that the rapidity by which cell lethality is produced by c9Ado may be related to inhibition of translation via its incorporation into RNA.

Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis.

A general procedure for detecting tyrosine kinase activity in crude or purified preparations using nondenaturing gel electrophoresis is presented. Samples are resolved by electrophoresis in minigels which are then incubated in an assay mixture containing [gamma-32P]ATP, poly(glutamic acid, tyrosine)4:1, and cofactors. Subsequently, gels are fixed and washed in trichloroacetic acid-pyrophosphate, dried, and analyzed by autoradiography or liquid scintillation counting. The procedure is simple and fast and allows analysis of different molecular weight forms of tyrosine kinase under linear kinetics at 37 degrees C without interference from phosphatases and proteases.

Appearance of membrane-bound tyrosine kinase during differentiation of HL-60 leukemia cells by immune interferon and tumor necrosis factor.

The effect of immune interferon (IFN-gamma) and recombinant tumor necrosis factor (rTNF-alpha) on cellular differentiation was investigated in human promyelocytic leukemia cell line HL-60. Both IFN-gamma and rTNF-alpha induced the appearance of the monocytic phenotype in a dose- and time-dependent manner as assessed by morphology, reduction of nitroblue tetrazolium and the induction of alpha-naphthyl butyrate esterase. Utilizing a nondenaturing polyacrylamide electrophoretic assay, it was revealed that a membrane-bound tyrosine kinase activity accompanied the appearance of the differentiated cell type. These results suggest that the induction of membrane-bound tyrosine kinase activity by IFN-gamma and rTNF-alpha may be an important characteristic of monocytic differentiation.

Effect of neplanocin A on differentiation, nucleic acid methylation, and c-myc mRNA expression in human promyelocytic leukemia cells.

The effect of the cyclopentenyl adenosine analog neplanocin A (NPC) on cell growth and differentiation was examined in the human promyelocytic leukemia cell line HL-60. Continuous exposure of HL-60 cells to 0.1-3.3 microM NPC resulted in a progressive reduction in cell growth which was accompanied by an increase in differentiation to cells with a myelocyte and neutrophil morphology. The latter effect was expressed as an increase in the capacity of cells to reduce nitro blue tetrazolium and reached a level of 40% of the total cell population. Preceding the phenotypic changes was the preferential inhibition of RNA and DNA methylation in comparison to inhibition of their synthesis which coincided with the formation of a metabolite of NPC with the chromatographic characteristics of S-adenosyl-L-methionine (AdoMet). In addition, c-myc mRNA expression, which is amplified in HL-60 cells, was markedly reduced following NPC treatment. These results indicate that NPC is an effective inhibitor of RNA and DNA methylation resulting from its conversion to an analog of AdoMet, and that these effects appear to be responsible for reduced c-myc RNA expression and the induction of myeloid differentiation in this cell line.

Synergistic effect of retinoic acid and calcium ionophore A23187 on differentiation, c-myc expression, and membrane tyrosine kinase activity in human promyelocytic leukemia cell line HL-60.

The effect of the combination of retinoic acid (RA) and calcium ionophore A23187 on cellular differentiation was assessed in promyelocytic leukemia cell line HL-60. RA (10(-10)-2.5 X 10(-8) M) or A23187 (4 X 10(-7) M) alone produced 15-22% differentiated cells as assessed by nitroblue tetrazolium reduction. Exposure of cells for 48 hr to the combination of 4 X 10(-7) M A23187 and 10(-10)-2.5 X 10(-8) M RA resulted in 20-86% of the cells capable of reducing nitroblue tetrazolium, but with no measurable level of nonspecific esterase activity. The combination of A23187 and either dimethyl sulfoxide, 1,25-dihydroxyvitamin D3, or immune interferon failed to produce a synergistic effect on differentiation. Addition of either the calmodulin antagonists, N-(6-aminohexyl)-5-chloronaphthalenesulfonamide and trifluoperazine, or the protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, during treatment with A23187 and RA did not block differentiation. Membrane tyrosine kinase activity was measured in cells treated with A23187 and RA in a nondenaturing gel system using the exogenous substrate poly(Glu:Tyr). Membrane-bound tyrosine kinase activity was not present in untreated or RA-treated cells but was induced by A23187 treatment alone and was markedly increased in cells 48 hr after treatment with the combination of A23187 and RA. Significantly greater reduction in c-myc mRNA levels was also observed 24 hr after treatment with RA and A23187 in comparison to that observed with either agent alone. These results suggest that a Ca2+-mediated process sensitizes cells to the differentiating effect of RA and that this effect is associated with a significant reduction of c-myc expression and the induction of membrane tyrosine kinase activity in this cell line.

3-Deazaneplanocin: a new and potent inhibitor of S-adenosylhomocysteine hydrolase and its effects on human promyelocytic leukemia cell line HL-60.

3-Deazaneplanocin, a new carbocyclic analog of adenosine, was synthesized as an inhibitor of S-adenosylhomocysteine hydrolase. The Ki of 3-deazaneplanocin for a purified hamster liver preparation of S-adenosylhomocysteine hydrolase was 5 X 10(-11) M, making this inhibitor 250-fold more potent than the previously known most potent inhibitor of this enzyme, 3-deazaaristeromycin. Inhibition was competitive with the substrate adenosine. Human promyelocytic leukemia (HL-60) cells treated with 10(-5) M 3-deazaneplanocin showed a pronounced elevation in S-adenosylhomocysteine which was 4-fold greater than that produced by an equimolar concentration of 3-deazaaristeromycim. This effect preceded a moderate reduction in cell growth and viability following continuous exposure for 6 days. Cellular differentiation as monitored by the reduction of nitroblue tetrazolium was not markedly affected except after 4 days exposure to 10(-5) M 3-deazaneplanocin where 60% of the viable cells were positive. These results indicate that 3-deazaneplanocin may have therapeutic potential as an anticancer or antiviral drug.