Cell killing, DNA polymerase inactivation and radiosensitization to low dose rate irradiation by mild hyperthermia in four human cell lines.

Four human cell lines (one fibroblast, two melanoma and one glioma) were evaluated for their responses to hyperthermia and thermalradiosensitization. For mild hyperthermia (40-42 degrees C), there was little to no chronic thermotolerance development during protracted heating for up to 72 h. In addition, there was no significant thermotolerance for polymerase inactivation during mild hyperthermia. For high temperature hyperthermia, polymerase beta was more thermal sensitive than aphidicolin sensitive polymerase alpha + delta + epsilon, (termed polymerase alpha) but during mild hyperthermia ther relative sensitivities were reversed. Polymerase beta was resistant to mild hyperthermia and polymerase alpha was very sensitive. Within each cell line there was a correlation between polymerase alpha inactivation and the degree of radiosensitization (TER) and amongst the cell lines the most radiation resistant cell line had less polymerase alpha inactivation than the most sensitive cell line for similar values of TER's. These data indicate that, amongst the cell lines, radiosensitivity and polymerase alpha sensitivity may influence TER and that for a given cell line, or possibly tumour, polymerase alpha inactivation may have potential as an indicator to determine TER for mild hyperthermia treatments in radiosensitization to low dose rates.

Differential inhibition of reverse transcriptase and cellular DNA polymerase-alpha activities by lignans isolated from Chinese herbs, Phyllanthus myrtifolius Moon, and tannins from Lonicera japonica Thunb and Castanopsis hystrix.

Two lignans, phyllamycin B and retrojusticidin B isolated from Phyllanthus myrtifolius Moon have been demonstrated to have a strong inhibitory effect on human immunodeficiency virus-1 reverse transcriptase activity (HIV-1 RT), but much less inhibitory effect on human DNA polymerase-alpha (HDNAP-alpha) activity. Fifty percent inhibitory concentrations of phyllamycin B and retrojusticidin B were determined to be 3.5 and 5.5 microM for HIV-1 RT, and 289 and 989 microM for HDNAP-alpha, respectively. The mode of inhibition was found to be non-competitive inhibition with respect to template-primer and triphosphate substrate. Several tannins such as caffeoylquinates (CQs) isolated from Lonicera japonica Thunb, galloylquinates (GQs) and galloylshikimates (GSs) purified from Castanopsis hystrix were shown to have a much less selective inhibitory effect on HIV-1 RT.

Effect of protracted mild hyperthermia on polymerase activity in a human melanoma cell line.

Radioresistant human melanoma SkMel-3 was evaluated with its sensitivity to thermal cell killing and polymerase inactivation. Cells were heated from 40 to 45 degrees C and demonstrated no thermal tolerance development for any of the temperatures tested. In addition, at 45 degrees C the heat survival curve showed a large shoulder indicating capacity for accumulation of sublethal heat damage. Also at 45 degrees C heating polymerase beta was more sensitive than polymerase alpha + delta + epsilon. At 42 degrees C, the polymerase sensitivities were nearly the same but at the lower temperatures (41 and 40 degrees C) polymerase beta became progressively more resistant than the polymerase alpha + delta + epsilon. Thus, mild hyperthermia effects may be different than high temperature hyperthermia and may be related to polymerase alpha + delta + epsilon activity.

[Template-primer-dependent inactivation of DNA polymerase alpha from human placenta by 2',3'-epoxyadenosine-5'-triphosphate]. / Matritsa-praimerzavisimaia inaktivatsiia DNK-polimerazy alpha iz platsenty cheloveka 2',3'-époksiadenozin-5'-trifosfatom.

Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase.

Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases.

The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.

Synthesis, cell growth inhibition, and antitumor screening of 2-(p-n-butylanilino)purines and their nucleoside analogues.

Derivatives of N2-(p-n-butylphenyl)guanine (BuPG) and 2-(p-n-butylanilino)adenine (BuAA) were synthesized and tested as inhibitors of mammalian DNA polymerase alpha, cell growth, and macromolecule synthesis. 2-(p-n-Butylanilino)-6-chloropurine (BuACl) served as a useful intermediate to prepare a series of 6-substituted analogues. BuACl, as its sodium salt, reacted with 2-deoxy-3,5-di-p-toluoyl-beta-D-ribofuranosyl chloride in acetonitrile to give 64% of the corresponding 9-beta nucleoside (blocked BuAdCl) and only 14% of the 7-beta isomer. Deblocking and substitution of chlorine in BuAdCl generated a series of 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)purine derivatives. Reaction of the sodium salt of BuACl with (2-acetoxyethoxy)methyl bromide also afforded, after deblocking and substitution of the 6-chloro group, a series of 2-(p-n-butylanilino)-9-[(2-hydroxyethoxy)methyl]purines. The bases synthesized were inhibitors of DNA polymerase alpha isolated from Chinese hamster ovary cells, the most potent compounds being 6-methoxy and 6-methylthio derivatives of 2-(p-n-butylanilino)purine. When tested for their ability to inhibit [3H]thymidine incorporation into DNA in HeLa cell cultures and the growth of exponentially growing HeLa cells, 9-(2-deoxy-beta-D-ribofuranosyl) derivatives had greater potency than their base counterparts, but "adenine" analogues, such as 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA, IC50 = 1 microM), were considerably more potent than N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG, IC50 = 25 microM). Derivatives bearing the 9-[(2-hydroxyethoxy)methyl] group were nearly as potent inhibitors of [3H]thymidine incorporation in these experiments as the corresponding deoxyribonucleosides. Base and deoxynucleoside derivatives also inhibited cellular RNA synthesis, and several compounds, at high concentrations, inhibited protein synthesis. BuPG, BuAA, and four deoxyribonucleoside derivatives of 2-(p-n-butylanilino)purines were tested against P-388 lymphocytic leukemia in mice. None of the compounds increased the survival time of test animals, but two of them, BuAdA and its 6-desamino derivative BuAdP, were lethal at the highest concentration used (400 mg/kg).

Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides.

2-(p-n-Butylanilino)adenine (BuAA), an homolog of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n-butylphenyl)guanine (BuPG), was transformed to its 2'-deoxyribonucleoside, BuAdA, and the corresponding 2'-deoxyribonucleoside 5'-phosphates, BuAdAMP, BuAdADP, and BuAdATP. All five forms of BuAA are highly selective inhibitors of mammalian pol alpha, and the action of each is subject to specific competitive antagonism by dATP. BuAdADP, and BuAdATP, like the corresponding forms of BuPG, are very potent pol alpha inhibitors, displaying apparent Ki's of less than 3 nanomolar on natural activated templates. BuAdATP, like BuPdGTP, also inhibits pol alpha-catalysed reactions directed by non-complementary, thymine-deficient templates, and it does so via a mechanism subject to specific antagonism by its natural homolog, dATP. The results of the BuAdATP-homopolymer experiments complement those of analogous experiments with BuPdGTP and the dCTP-specific pol alpha inhibitor, aphidicolin, and strengthen the suggestion that mammalian pol alpha contains dNDP and dNTP binding sites which can recognize specific bases without direction by templates.

Effects of sodium selenite and selenate on DNA and RNA synthesis in vitro.

The effects of sodium selenite and sodium selenate on DNA and RNA synthesis have been examined using intact HeLa cells, isolated nuclei and extracted polymerases. Selenate had no effect on any of the systems examined. Selenite inhibited DNA synthesis in intact cells and in isolated nuclei, and to a limited extent also inhibited DNA polymerase alpha. Selenite also inhibited RNA synthesis in intact cells and alpha-amanitin resistant RNA synthesis in isolated nuclei (i.e., synthesis catalyzed by RNA polymerase I and III). It had no effect on alpha-amanitin sensitive synthesis (catalyzed by RNA polymerase II) at concentrations up to 500 microM. However, transcription of exogenous DNA by extracted RNA polymerase II (as well as by polymerase I and III) was inhibited by selenite.

The effect of hydroxycamptothecin in the activity of RNA and DNA polymerases prepared from murine hepatoma cells.

Hydroxycamptothecin (HCPT) is an antitumor alkaloid isolated from Camptotheca acuminata indigenous to China. It could reduce the activity of nuclear RNA polymerase II and I(III) of hepatoma cells. HCPT at 25-100 microM caused a remarkable inhibition on DNA polymerase alpha whilst only a slight inhibition on beta. The inhibitory action on alpha was restored by increasing amounts of enzyme or DNA template, but unchanged by varying amounts of substrate. It is suggested that HCPT may exert a stronger inhibition on DNA replication process.

Inhibition of DNA polymerase from L1210 murine leukemia by a sulfhydryl reagent from agaricus bisporus.

The 490 quinone, a natural sulfhydryl-arylating reagent from the mushroom, Agaricus bisporus, markedly inhibited L1210 murine leukemia DNA polymerase alpha while resulting in little inhibition of DNA polymerase beta from this source. This quinone was more strongly inhibitory than p-chloromercuri-benzoate or N-ethylmaleimide and was less readily neutralized by sulfhydryl-containing molecules such as dithioerythritol. Preliminary experiments indicate that DNA protects DNA polymerase alpha from inhibition by the 490 quinone. The inhibition of DNA synthesis by quinone 490 may contribute significantly to the cytotoxicity of this compound and to the potential of gamma-L-glutaminyl-4-hydroxybenzene as an antitumor agent.