Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Enhancement of prostaglandin synthesizing activity by the treatment with sodium n-butyrate on cloned mastocytoma cells and various other tissue cell lines

We have compared the effects of n-butyrate on the prostaglandin synthesizing activities of cloned mouse mastocytoma cells, and various other tissue culture cell lines. Cells were treated with 1 mM n-butyrate for 40 hrs before harvesting. Prostaglandin synthesizing activities of the treated and the control cells were examined in a cell-free assay system. The treatment of some of the cloned mastocytoma cells with n-butyrate brought about the synthesis of prostaglandin D2, E2 and F2 alpha that were not synthesized by the control cells. The treatment of epithelial liver cells (BC-90) also resulted in the formation of 6-keto-prostaglandin F1 alpha which was not formed by the control cells. However, n-butyrate caused relatively small changes in the prostaglandin synthesizing activities of other clones of mastocytoma cells, mouse hepatoma cells, HeLa cells, rat granuloma cells and human embryonic fibroblasts. These data suggest differential effects of n-butyrate on different types of cultured cells.     

Sodium n-butyrate induces prostaglandin synthetase activity in mastocytoma P-815 cells

Cultured mouse mastocytoma P-815 cells were treated with 1 mM sodium n-butyrate for 40 h. The treated cell homogenate showed high activities in synthesizing prostaglandin D2, E2, and F2 alpha. Such activities were virtually absent in untreated cell homogenate. Direct addition of sodium n-butyrate to the homogenate showed no effects. Pre-exposure of cells to acetylsalicylic acid did not diminish the effect of the subsequent treatment with sodium n-butyrate. These data suggest that sodium n-butyrate induces fatty acid cyclooxygenase in P-815 cells.     

Control of DNA polymerase alpha, beta and gamma activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.     

Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production.

We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3. HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did. Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ. Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures. Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found. Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta. The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective. In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures. HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures. Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells. In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells. Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells. These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures.     

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.     

A DNA template recognition protein: partial purification from mouse liver and stimulation of DNA polymerase alpha

A protein that specifically enhances up to 13-fold the rate of copying of poly(dT) template by DNA polymerase alpha was partially purified from chromatin of regenerating mouse liver cells. This stimulatory protein, designated herein factor D, also increases 2-3-fold the activity of polymerase alpha with heat-denatured DNA and with primed, circular single-stranded phi X174 DNA. However, factor D has no detectable effect on the copying by polymerase alpha of poly(dG), poly(dA), and poly(dC) templates. Activity of mouse DNA polymerase beta is not affected by factor D with all the tested templates. In contrast to polymerase alpha, factor D is resistant to inactivation by N-ethylmaleimide and calcium ions, but it is readily heat-inactivated at 46 degrees C and is inactivated by trypsin digestion. Partially purified factor D is not associated with detectable activities of DNA polymerase, DNA primase, deoxyribonucleotidyl terminal transferase, and endo- or exodeoxyribonuclease.     

Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation. The purified enzyme proved to be a monomeric protein with a molecular weight of about 30,500. The enzyme catalyzed not only the reduction of the 11-keto group of prostaglandin D2 but also the reduction of 9,11-endoperoxide of prostaglandin H2 and various carbonyl compounds (e.g. phenanthrenequinone). Experiments using column chromatography, polyacrylamide gel electrophoreses, immunotitration using antibody against the purified enzyme, and heat treatment indicated that three enzyme activities resided in a single protein. Although phenanthrenequinone and prostaglandin D2 competitively inhibited the prostaglandin D2 and phenanthrenequinone reductase activities, respectively, these two substrates were all but ineffective on the prostaglandin H2 (at the Km value) reductase activity up to 14-fold of those Km values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin D2, prostaglandin H2, and various carbonyl compounds and that prostaglandin D2 and prostaglandin H2 are metabolized at two different active sites, yielding prostaglandin F2 alpha as the reaction product.     

Enhancement of prostaglandin synthesizing activity by the treatment with sodium n-butyrate on cloned mastocytoma cells and various other tissue cell lines

We have compared the effects of n-butyrate on the prostaglandin synthesizing activities of cloned mouse mastocytoma cells, and various other tissue culture cell lines. Cells were treated with 1 mM n-butyrate for 40 hrs before harvesting. Prostaglandin synthesizing activities of the treated and the control cells were examined in a cell-free assay system. The treatment of some of the cloned mastocytoma cells with n-butyrate brought about the synthesis of prostaglandin D₂, E₂ and F_2α that were not synthesized by the control cells. The treatment of epithelial liver cells (BC-90) also resulted in the formation of 6-keto-prostaglandin F_1α which was not formed by the control cells. However, n-butyrate caused relatively small changes in the prostaglandin synthesizing activities of other clones of mastocytoma cells, mouse hepatoma cells, HeLa cells, rat granuloma cells and human embryonic fibroblasts. These data suggest differential effects of n-butyrate on different types of cultured cells.     

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.     

Appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells during monocytic differentiation: enhancement of thromboxane synthesis by 1 alpha,25-dihydroxyvitamin D-3

The appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells was investigated during 1 alpha,25-dihydroxyvitamin D-3-induced monocytic differentiation. 1 alpha,25-Dihydroxyvitamin D-3-treated HL-60 cells acquired the ability to convert [1-14C]arachidonic acid to thromboxane B2 and prostaglandin E2 during monocytic differentiation. The major cyclooxygenase product synthesized by the HL-60 cells after 3-4 days exposure to 1 alpha,25- dihydroxyvitamin D-3 (48 nM) was thromboxane B2 and its production was about 19-25-times higher than that of untreated HL-60 cells. The percent conversion of thromboxane B2 from [1-14C]arachidonic acid in the 1 alpha,25-dihydroxyvitamin D-3 (48 nM, 3 day exposure)-treated HL-60 cells was about 4.4% as compared to that (about 0.3%) of the untreated cells, whereas the percent conversion of thromboxane B2 from [1-14C]prostaglandin H2 in the 1 alpha,25-dihydroxyvitamin D-3-treated cell homogenate was about 22.4% as compared to that (about 13.6%) of the untreated cell homogenate. The stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on thromboxane B2 production from [1-14C]arachidonic acid and from [1-14C]prostaglandin H2 in HL-60 cells was inhibited by the addition of cycloheximide (1 microgram/ml). However, 1 alpha,25-dihydroxyvitamin D-3 (48 nM) did not significantly stimulate the arachidonic acid release either in HL-60 cells or in 1 alpha,25-dihydroxyvitamin D-3-induced cells. These results suggest that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on the thromboxane production in HL-60 cells was not due to the activation of phospholipase A2 but due to the induction of fatty acid cyclooxygenase and thromboxane synthetase activities. Thromboxane A2 actively produced during the monocytic differentiation of HL-60 cells could influence the cell adhesiveness of the monocyte-macrophage-differentiated cells.     

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase alpha activity

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.     

The interrelation between DNA synthesis rates and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells

Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.     

Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.     

The effect of aphidicolin on DNA synthesis in isolated HeLa cell nuclei.

The effect of the inhibitor aphidicolin on DNA synthesis in isolated nuclei from HeLa cells and on the activities of partially purified DNA polymerases has been tested. Aphidicolin inhibited DNA synthesis and DNA polymerase alpha very efficiently whereas DNA polymerases beta and gamma were insensitive to the drug. The results indicate that DNA polymerase alpha is the polymerase active during elongation as well as in the gapfilling process of discontinuous DNA synthesis.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.