Human N-myristoyltransferase amino-terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction.

N-Myristoyltransferase (NMT) catalyzes the cotranslational acylation with myristic acid of the NH2-terminal glycines of a number of cellular and viral proteins. Most of the in vitro NMT activity (60-85%) in isoosmotic cell homogenates of human lymphoblastic leukemia (i.e. CEM and MOLT-4) and cervical carcinoma (i.e. HeLa) cells was shown to be associated with the ribosomal subcellular fractions by differential centrifugation. Also found in the ribosomal fractions was a approximately 60-kDa protein that was specifically immunoblotted with an anti-human NMT (hNMT) peptide antibody. This approximately 60-kDa protein was stable in the presence of proteolytic enzyme inhibitors but was gradually converted into a approximately 46-kDa species when stored in the absence of protease inhibitors. Sucrose density gradient centrifugation of the ribosomal fraction resulted in the hNMT activity sedimenting exactly coincident with the 260 nm absorption profile and exhibiting A260/A280 absorption ratios >1.8, indicating an association of NMT with putative ribosomal particle(s)/subunit(s). The subcellular targeting of hNMT was also examined by immunoblotting subcellular fractions from HeLa cells transfected with plasmids containing FLAG epitope-tagged hNMT inserts corresponding either to the originally assigned hNMT gene or to an alternative open reading frame initiated from an in-frame start site upstream from the assumed hNMT start site. Anti-FLAG immunoblotting of cells transfected with a plasmid containing the larger insert revealed FLAG-NMT primarily in the ribosomal fraction with an apparent molecular mass similar to the approximately 60-kDa native hNMT. In contrast, immunoblotting of cells transfected with a plasmid containing the smaller insert identified a approximately 50-kDa FLAG-NMT predominantly in the cytosolic fraction. An analysis of mixtures of CEM ribosomes and serial dilutions of purified recombinant FLAG-NMTs demonstrated that the approximately 60-kDa FLAG-NMT binds ribosomes with higher affinity than the approximately 50-kDa FLAG-NMT. These in vivo and in vitro subcellular targeting and recombinant expression experiments identify a native hNMT that is 10-12 kDa larger than the enzyme predicted by the originally assigned hNMT gene and which is apparently translated from an alternative up-stream start site. The data also indicate that although the unique NH2-terminal residues encoded by this larger open reading frame are not required for in vitro catalytic activity, they do provide signal(s) involved in targeting hNMT to the ribosomal subcellular fraction where cotranslational N-myristoylation occurs.

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C.

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.

Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells.

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.

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.

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.

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.

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.

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.

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.

In vitro translational activity of messenger RNA following treatment of human colon carcinoma cells with sangivamycin.

Total mRNA from human colon carcinoma cell line HT-29 treated with the pyrrolopyrimidine antibiotic, sangivamycin(7-deaza-7-carboxamidoadenosine), was assessed in vitro using a reticulocyte lysate translation system. Under conditions of known drug-induced cell lethality, sangivamycin-modified mRNA showed a diminished translational capacity. The decreased activity of drug-modified mRNA increased in proportion to the time-dependent cytocidal effects of sangivamycin in this cell line. These data suggest that the lethal effects of this drug may be associated, in part, with a reduced ability of drug-modified mRNA to sustain translation in situ.

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.

In vitro translation of messenger RNA following exposure of human colon carcinoma cells in culture to 5-fluorouracil and 5-fluorouridine.

Total mRNA from human colon carcinoma cell line HT-29 treated with either 5-fluorouracil or 5-fluorouridine was assessed in vitro in a reticulocyte lysate translation system. Under conditions of known drug-induced cell lethality, fluoropyrimidine-modified mRNA did not show major quantitative or qualitative changes in translational activity. These results suggest that drug-modified mRNA is probably not associated with the cytotoxicity manifested by these drugs.

Cytokinetic and biochemical effects of 5-iminodaunorubicin in human colon carcinoma in culture.

The semisynthetic anthracycline, 5-iminodaunorubicin (IM), was investigated to see whether modification of the benzoquinone moiety to produce a drug with low free radical potential would alter the cytotoxic and biochemical characteristics of this drug in comparison to Adriamycin (ADR), an agent with high free radical potential. Cell viability was measured in human colon carcinoma (HT-29) cells by soft-agar cloning. Upon exposure to either log-phase or early plateau-phase cells for 2 hr to IM or ADR, a threshold exponential cell lethality curve was obtained. Prolonging during exposure to 24 hr produced an exponential decline in cell survival and a marked reduction in viability of both log-phase and early plateau-phase cells. Inhibition of DNA synthesis in log-phase cells after 2 and 24 hr of exposure to IM and ADR paralleled the increased cell lethality produced by the drugs. In contrast, total RNA synthesis was not inhibited by IM, whereas ADR impaired both RNA and DNA synthesis. Nuclear rRNA, synthesis was not significantly inhibited following 24 hr of exposure to 10(-7) M ADR or IM but was inhibited by 85 and 35% at 10(-6) M ADR or IM, respectively. The affinity of IM and ADR for HT-29 DNA was measured in vitro by displacement of acridine orange binding and was found to be similar for both analogs. These studies suggest that the cytotoxicity of IM and ADR results from the interactions of these drugs with DNA.

Lymphatic absorption and tissue disposition of liposome-entrapped [14C]adriamycin following intraperitoneal administration to rats.

The lymphatic absorption and tissue distribution of free [14C]Adriamycin, "empty" [3H]liposomes, free [14C]Adriamycin plus empty [3H]liposomes, and [14C]Adriamycin entrapped into [3H]liposomes have been examined at intervals after i.p. injection into rats. Following treatment with empty [3H]liposomes, almost 30% of the liposomal lipid marker was recovered in 24-hr thoracic duct lymph, but when [14C]Adriamycin was added to or encapsulated in liposomes, this value was reduced to 10%. Conversely, only 1% of free [14C]Adriamycin was recovered in 24-hr lymph, but liposomal encapsulation produced a six-fold increase in this value. Studies on the tissue distribution of the liposomal lipid marker after dosing with empty liposomes revealed uptake by diaphragm, liver and spleen, but the highest tissue concentrations were noted in lymph nodes. Liposomal encapsulation of Adriamycin altered its tissue disposition, chiefly by increasing the concentration of drug equivalents in diaphragm, liver and spleen. Although free Adriamycin was accumulated by lymph nodes to some extent, this lymph node accumulation was markedly enhanced by liposomal encapsulation and was present only in those nodes through which lymph draining the peritoneal cavity passes. This finding, together with the observation that diaphragm and thoracic duct lymph contain relatively high levels of liposomal lipid and Adriamycin equivalents, indicates that liposomes are selectively absorbed from the peritoneal cavity by lymphatics and are retained by certain lymph nodes. The results of this study suggest that i.p. administration of liposome-encapsulated drugs may provide a means of selectively concentrating anti-tumor agents in lymphatic channels and lymph nodes.