Isolation and characterization of nuclear RNA polymerase II from chicken myeloblastosis cells.

Nuclear RNA polymerases of chicken myeloblastosis cells were solubilized and fractionated by diethylaminoethyl-Sephades A25 column chromatography. Both alpha-amanitin-insenstitive (polymerase I) and- sensitive (polymerase II) species were isolated. Polymerase activity, contained two peaks of enzyme (IIa and IIb), which were further purified by glycerol gradient centrifugation. The partially purified enzymes were characterized by their requirement of four nucleoside triphosphates and metal ions and by their sensitivity to several inhibitors. The enzymes were compared with RNA polymearases derived from normal chickent bone marrow cells,and the total extractable myeloblastosis than in bone marrow cells. Polymearse II from both cell types was shown to be sensitive to cytosine arabinoside triphosphate inhibiton.

Nucleoside uptake and membrane fluidity studies on N-trifluoroacetyladriamycin-14-O-hemiadipate-treated human leukemia and lymphoma cells.

N-Trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a DNA nonbinding derivative of Adriamycin, was studied for its effect on the uptake of labeled nucleosides into human leukemia (ML-1) and lymphoma (P3HR-1) cells in culture. After preincubation with AD 143 at concentrations as low as 5.2 microM (ML-1) or 13 microM (P3HR-1), the ability of the cells to take up extracellular labeled nucleosides was decreased by more than 50%. Similar experiments with Escherichia coli cells showed that AD 143 at the same concentrations did not have any effect. Influx of [3H]thymidine or [3H]uridine was studied by centrifugation of the cells through phthalate oil mixture, and it was found that the influx of the labeled nucleosides was decreased after treatment of the cells with AD 143. An increase in the membrane fluidity was observed after treatment of the cells with AD 143, as revealed by electron paramagnetic resonance spectroscopy studies. These observations suggest that the decreased incorporation of [3H]thymidine and [3H]uridine into acid-precipitable material that we observed earlier in the AD 143-treated cells may in part be the result of the AD 143-induced alteration of cell membrane activities, which in turn causes an inhibition of nucleoside uptake.

Inhibition of human lymphoma DNA-dependent RNA polymerase activity by 6-mercaptopurine ribonucleoside triphosphate.

6-Mercaptopurine was found to inhibit the growth of cultured human lymphoma P3HR-1 cells and the incorporation of [3H]-uridine into trichloroacetic acid-precipitable materials of the cells. One of the derivatives of 6-mercaptopurine, 6-mercaptopurine ribonucleoside triphosphate (6-thio-ITP), was found to inhibit in vitro RNA synthesis (both engaged and free enzyme activities) of the isolated nuclei from P3HR-1 cells. The alpha-amanitin-resistant RNA polymerase (polymerase I) and alpha-amanitin-sensitive RNA polymerase (polymerase II) of the cells were isolated and partially purified by either diethylaminoethyl cellulose or diethylaminoethyl Sephadex column chromatography, followed by DNA-cellulose affinity chromatography. It was found that these partially purified enzymes were also sensitive to 6-thio-ITP inhibition. Kinetic studies showed that the inhibition of RNA polymerase activities by 6-thio-ITP could be reversed by increasing concentrations of guanosine 5'-triphosphate in the reaction mixture, indicating that 6-thio-ITP may act as a competitive inhibitor of the enzymes by competing with guanosine 5'-triphosphate for its enzyme-binding site. These data suggest that inhibition of RNA transcription by 6-thio-ITP may be considered as one of the mechanisms of the cytotoxic action of 6-mercaptopurine in human tumor cells.

Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro.

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.

The 24,000 Da subunit is not required for the RNA synthesis activity of chicken leukemia RNA polymerase II.

The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.

Interaction of N-trifluoroacetyladriamycin-14-O-hemiadipate with chicken leukemia RNA polymerase. Formation of drug-enzyme complex.

The biochemical mechanism of the N-trifluoroacetyladriamycin-14-O-hemiadipate-induced inhibition of RNA synthesis in vitro by chicken (myeloblastosis) leukemia RNA polymerase II was studied. The inhibition was found to be dependent upon preincubation of the drug with the enzyme prior to enzyme assays, suggesting that drug-enzyme interactions occur. A drug-enzyme association complex was subsequently isolated through glycerol gradient sedimentation and further characterized by fluorescent microscopic studies. The drug was dissociated from the complex upon sodium dodecyl sulfate (SDS)-gel electrophoresis, revealing the non-covalent nature of the binding between the drug and the RNA polymerase.

Activation of human leukemia protein kinase C by tumor promoters and its inhibition by N-trifluoroacetyladriamycin-14-valerate (AD 32).

N-Trifluoroacetyladriamycin-14-valerate (AD 32), a lipophilic, DNA non-binding analog of Adriamycin (ADR), was found to be a potent inhibitor of the membrane-bound enzyme, protein kinase C (PKC). PKC was isolated and purified from human leukemia ML-1 cells, and the enzyme activity was shown to be activated by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). AD 32, nevertheless, inhibited the activation of PKC by TPA or PDBu. The IC50 values for AD 32 inhibition of PKC activation were 0.85 microM for TPA and 1.25 microM for PDBu. Under the same assay conditions, ADR demonstrated much higher IC50 values: 550 microM for TPA and greater than 350 microM for PDBu. The inhibition of PKC by AD 32 was further shown to be competitive in nature; AD 32 inhibited the binding of [3H]PDBu to PKC. Therefore, AD 32 competes with the tumor promoter for the PKC binding site and prevents the latter from both interacting with the phospholipid and binding to PKC. These effects of AD 32 were reproduced in situ; incubation of human leukemia ML-1 cells with TPA showed an increased phosphorylation of cellular proteins, and the TPA-induced protein phosphorylation was inhibited by the addition of AD 32 to the cultured cells.

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe.

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.

Colony hybridizations using nylon membranes and RNA probes--an improved method for screening bacterial clones containing bluetongue virus genome.

Bluetongue virus (BTV) total genomic and isolated individual segment dsRNAs end-labeled with 32P were successfully used as probes in colony hybridization to detect clones of BTV genomic material. The RNA probes were highly specific for cloned BTV genomic material. DNA probes, however, gave false positive results. DNA from bacterial clones was fixed to nylon and nitrocellulose membranes. The hybridized nylon membranes could be stripped of probe and reprobed at least 6 times without loss of signal strength.

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Detection of simian immunodeficiency virus RNA from infected rhesus macaques by the polymerase chain reaction.

A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIVmac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 x 10(5) PBMC of two animals.

Kappa-opioid receptors on lymphocytes of a human lymphocytic cell line: morphine-induced up-regulation as evidenced by competitive RT-PCR and indirect immunofluorescence.

We have previously shown that classical brain-like kappa opioid receptors (KOR) are constitutively expressed in lymphocytic cells. including human CEM x174 T-B hybrid cells, Jurkat -T4 cells, human peripheral blood mononuclear cells (PBMC), human CD4+ cells and monkey PBMC (Biochem. Biophys. Res. Commun. 209 (1995) 1003). The present study further demonstrates that the KOR of lymphocytes are activated in the presence of extracellular morphine or U50,488H, a KOR selective agonist, and the activation causes an increase in the expression of KOR mRNA, as determined by a quantitative competitive Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) procedure. The observed agonist-induced KOR up-regulation was blocked by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-binaltorphimine (a KOR-selective antagonist). Up-regulation of lymphocytic KOR by morphine was also evidenced by flow cytometric analysis of phycoerythrin (PE) amplification of fluorescein isothiocyanate-conjugated arylacetamide labeling of the KOR. Although morphine binds primarily to mu-opioid receptors, together with the previously reported phenomenon that morphine modulation of immune functions also exists in mu-opioid receptor knockout mice, the present study confirms that opioids such as morphine may exert their effects through multiple opioid receptor types and that the effects of morphine or endogenous opioids on immune cells could not be simply adduced from the anticipated effects of a synthetic, selective opioid receptor ligand.

The effect of the insecticide heptachlor on ras proto-oncogene expression in human myeloblastic leukemia (ML-1) cells.

Expression of the ras proto-oncogene mRNA in human myeloblastic leukemia (ML-1) cells was analyzed as a function of cDNA amplification by polymerase chain reaction (PCR). By using a pair of oligonucleotides that flank exon-2 from opposite strands (5' and 3') of H-ras cDNA for PCR amplification, ML-1 cells were found to express a 112 bp segment of the ras transcript. A rapid decline in the expression of this transcript was seen in cells treated with heptachlor, a chlorinated hydrocarbon insecticide. Expression of the same ras segment was not affected by treatment of ML-1 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, addition of serum to quiescent, heptachlor-treated cultures of ML-1 cells inhibited the effect of heptachlor and restored the expression of the ras protooncogene mRNA.

Heptachlor and the mitogen-activated protein kinase module in human lymphocytes.

The organochlorine pesticide heptachlor constitutes a potential health hazard because of its persistence in nature, its reported contamination in food and milk, and its possible carcinogenic effects. As a tumor promoter, heptachlor induces human myeloblastic leukemia cells to differentiate, and also down-regulates the tumor suppressor gene p53 in human immune cells. In this study, the heptachlor signaling pathway in human lymphocytes was studied. Addition of heptachlor to human CEM x174 lymphocytic cells reduced the cellular levels of MAP kinase (MAPK, mitogen-activated protein kinase) cascade proteins, including ERK1 (a 44-kDa MAPK), ERK2 (a 42-kDa MAPK), a 85-kDa and a 54-kDa MAP kinase, MEK1 (a 45-kDa ERK kinase) and MEKK (a 78-kDa MEK kinase). However, heptachlor treatment caused a marked increase in the expression of the activated (Thr- and Tyr-dually phosphorylated) ERK1 and ERK2 in the cells. These studies indicate that mitogen-activated protein kinases are important intermediates in the signal transduction pathway of immune cells upon heptachlor exposure, and the observation of stimulation of activated MAP kinases without a simultaneous accumulation of basal enzymes may suggest the involvement of a negative feedback control mechanism in the pathway.

Cremophor EL inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced protein phosphorylation in human myeloblastic leukemia ML-1 cells.

Cremophor EL, a polyloxyethylene castor oil derivative used clinically as a parenteral vehicle, inhibits protein kinase c activity in vitro. The tumor promoting agent TPA (12-0-tetradecanoylphorbol-13-acetate) activated protein kinase C and induced phosphorylation of cellular proteins of human myeloblastic leukemia ML-1 cells. Polypeptides of 56 KDa, 44 KDa, 37 KDa, 35 KDa and 31 KDa were particularly phosphorylated in response to TPA activation. However, the phosphorylations of these polypeptides, especially that of 37 KDa, were greatly reduced by treatment of the TPA-activated ML-1 cells with Cremophor EL. Cremophor EL also inhibited the growth of ML-1 cells. On the other hand, the TPA-induced cell differentiation in ML-1, which is considered a separate event from protein kinase C activation, was not affected by Cremophor EL. These studies suggest biological implications for the observed in vitro activity of Cremophor EL. The studies may also provide a mechanism for the Cremophor EL-associated cytotoxicities observed when it is used clinically as a parenteral vehicle.

Adriamycin interacts with diacylglycerol to inhibit human leukemia protein kinase C.

Protein kinase C (PKC) from human leukemia ML-1 cells was found to be susceptible to inhibition by the antineoplastic anthracycline adriamycin (ADR). Half-maximal inhibition (IC50 value) was observed at 200 microM. However, preincubation of ADR with phosphatidylserine (PS) or PKC enzyme, prior to the enzyme assay, reduced the IC50 value from 200 microM to 52 microM or 40 microM, respectively, indicating an affinity of ADR for PS, and also a possible action site for ADR on PKC molecules. Preincubation of ADR with diacylglycerol (DAG) before the PKC assay resulted in a more pronounced effect, i.e., a more rapid decline of PKC activity with an IC50 value of 7 microM. However, the IC50 for ADR inhibition was not altered when ATP, histone or Ca++ were preincubated with ADR. Studies of the kinetic nature of the inhibition revealed that ADR inhibition assumes competitive kinetics with respect to DAG. Therefore, the mechanism by which ADR inhibits PKC activity may involve a multi-site action: a primary interaction with DAG, and a secondary lower interaction with membrane PS and PKC apoenzyme.

Mechanistic studies on N-benzyladriamycin-14-valerate (AD 198), a highly lipophilic alkyl adriamycin analog.

AD 198, a novel lipophilic N-alkyl derivative of adriamycin (ADR) and a potential anticancer agent for preclinical development, was studied for its effects on the activities of DNA and RNA polymerases in vitro and its ability to bind DNA. AD 198, which contains a benzyl substituent on the glycosidic amine, was found to interact with DNA through drug-DNA binding to an extent less than its parent compound ADR as shown by fluorescent emission spectra studies. It had a preferential inhibitory effect against RNA vs. DNA synthesis in vitro by RNA or DNA polymerases from both E. coli and chicken leukemia cells. Preincubation studies indicated that AD 198 may inhibit the activity of E. coli RNA polymerase through drug-template interaction and that of leukemic RNA polymerase, which uses single stranded DNA as template, through enzyme inactivation.

The effect of heptachlor, a chlorinated hydrocarbon insecticide, on p53 tumor suppressor in human lymphocytes.

Previous studies have shown that heptachlor, a chlorinated hydrocarbon insecticide, is a liver tumor promoter in rats and mice and induces tumor promoting-like alterations in human myeloblastic leukemia cells. The nature of tumor promotion is multifaceted and has recently been shown to include suppression of programmed cell death (apoptosis) as a mechanism by which a tumor promoter can prolong cell viability. The ability of tumor promoters to suppress apoptosis prompted us to address the question of whether heptachlor is capable of effecting the expression of genes involved in lymphocyte apoptosis, in particular, the p53 tumor suppressor gene. Experiments with a CEM x 174 cell line, a hybrid of human T and B cells, revealed that heptachlor downregulated p53 gene expression at the post-transcriptional level without changing levels of mRNA in the cells. The heptachlor-induced reduction in the basal levels of expression of this gene was both in a concentration and time-dependent manner.

Effect of the chlorinated hydrocarbons heptachlor, chlordane, and toxaphene on retinoblastoma tumor suppressor in human lymphocytes.

Organochlorine use over the past 50 years has resulted in the contamination of soil, water, plant and animal species. This contamination has created a long-lasting environmental problem, as the members of the organochlorine class of pesticides are resistant to degradation and have been labeled as persistent bioaccumulators. Studies have shown certain organochlorines to be tumor promoters, liver toxicants and to induce immune cell dysfunction in rats and mice. Our laboratory has shown that the organochlorines heptachlor and chlordane affect leukocytic gene expression and differentiation. In this study, experiments with CEM x 174 cells, a hybrid of human T and B cells, were performed to investigate the effects of the tumor promoter heptachlor and its congeners chlordane and toxaphene on retinoblastoma (Rb) gene expression. The results indicated that heptachlor, chlordane or toxaphene, in the range of 10-50 microM, were able to reduce Rb protein levels in a concentration-dependent manner. In the case of heptachlor, the reduction could be seen as early as 12 h and was time-dependent. Analysis of Rb mRNA levels revealed no detectable difference over the same concentration range. These results suggest that members of the organochlorine class are able to downregulate Rb expression at the post-transcriptional level, an effect similar to that on p53 tumor suppressor previously reported by our laboratory.

Suppression of chemokine-induced chemotaxis of monkey neutrophils and monocytes by chlorinated hydrocarbon insecticides.

Chemokines, characterized as pro-inflammatory chemicals made by the immune system, consist of a family of low molecular weight proteins with potent in vitro chemotactic activity causing leukocyte accumulation in vivo. This study determines the effects of organochlorine pesticide exposure on the chemotactic functions of monkey neutrophils and monocytes, using a 48-well chemotaxis chamber. Chemokines IL-8 (interleukin-8) and RANTES were used as the chemoattractants to induce chemotaxis among these monkey leukocytes. Monkey neutrophils or monocytes were first treated with heptachlor, chlordane or toxaphene for 1 hour at 37 degrees C, and the number of cells migrating toward 200 ng/ml IL-8 (for neutrophils) or 100 ng/ml RANTES (for monocytes) were scored. Inhibition of chemotaxis was seen with all samples after treatment with heptachlor, chlordane and toxaphene at concentrations from as low as 10(-14) M to 10(-5) M. Among the three compounds studied, toxaphene was the least effective in preventing monocytes from migrating toward RANTES. The ability of these pesticides to inhibit chemotaxis did not correlate directly with their potential apoptotic effects on the monkey leukocytes. These studies suggest that exposure to organochlorine pesticides may alter leukocyte-related immune functions.