The inhibition of DNA synthesis by cannabinoids.

Several of the cannabinoids found in marihuana have been shown to inhibit tumor growth and increase the life-span of mice bearing the Lewis lung adenocarcinoma. When trypsin-dispersed isolated Lewis lung cells are incubated in vitro, they maintain their capacity to carry out macromolecular synthesis (RNA, DNA, protein). This process can be inhibited by cytosine arabinoside, actinomycin D, or methyl-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, whereas cyclophosphamide, an agent that must be bioactivated, was inactive. Inhibition of DNA synthesis as measured by [3H]thymidine uptake into acid-insoluble material was used as an index of cannabinoid activity against isolated Lewis lung cells, L1210 leukemia cells, and bone marrow cells incubated in vitro delta9-, delta8-, 1-hydroxy-3-n pentyl-, and 1-delta8-tetrahydrocannabinol, and cannabinol demonstrated a dose-dependent inhibition of DNA synthesis whereas cannabidiol and 1-hydroxy-3-n-pentylcannabidiol were markedly less inhibitory in our in vitro cell systems. Furthermore, our in vitro observations with these cannabinoids are supported by in vivo tumor inhibition studies. Ring modifications as in cannabichromene or cannabicyclol abolish in vitro activity as does dihydroxylation at the 8beta and 11 positions of 1-delta9-trans-tetrahydrocannabinol. Delta9-trans-tetrahydrocannabinol demonstrated the least toxicity of all inhibitory cannabinoids in vivo; this is supported by its lesser effect on bone marrow DNA synthesis in vitro.     

The cellular pharmacology of oxaliplatin resistance

Oxaliplatin is a third generation platinum compound that differs from cisplatin and carboplatin in having a broader spectrum of antitumour activity. Molecular studies suggest that oxaliplatin adducts are recognised and processed differently than those produced by the earlier generation Pt-containing drugs. We report here studies on the kinetics of the development of oxaliplatin resistance, and the changes in the cellular pharmacology of oxaliplatin that accompany the emergence of the resistant phenotype in five parental human tumour cell lines and their sub-lines selected for acquired oxaliplatin resistance in vitro. During selection, resistance did not substantially increase until after at least six cycles of oxaliplatin treatment. Oxaliplatin demonstrated schedule-dependency with a 1-h exposure being substantially less cytotoxic than a continuous exposure. Whole cell uptake was linear with concentration, but uptake in the resistant cells averaged only 27+/-10 S.D.% of that in the sensitive cells. Pt accumulation in DNA was markedly reduced in four of the five resistant cell lines, but this did not correlate with either IC(50) or total cellular accumulation. Four of the five resistant sub-lines also demonstrated increased tolerance to adducts in DNA that ranged from 3.1 to 7.6-fold. We conclude that development of acquired resistance to oxaliplatin is accompanied by independent defects in both whole cell uptake and in adduct formation.     

1-beta-D-arabinofuranosylcytosine-induced chromatid breakage: effect of inhibition of DNA synthesis.

The frequency of chromatid breakage induced by 1-beta-D-arabinofuranosylcytosine was decreased when hamster cell fibroblasts were treated with chemical agents that interferred with DNA synthesis immediately before addition of the analog. This phenomenon occurred in both the S and G2 phases of the cell cycle.     

The role of suppression of DNA synthesis and inhibition of cell cycle progression in cellular sensitivity to alkylation damage

A u.v. sensitive Chinese hamster cell line V79/79 has been shownto be also more sensitive to methyl methanesulphonate (MMS)and nitrogen mustard (HN2) exposure than wild-type V79 cells.A comparison of the effects of the two alkylating agents onDNA synthesis measured by [³H]thymidine (TdR) incorporationinto whole cells and by alkaline sucrose gradient sedimentation¹⁴C-labelled template and of pulse labelled DNA revealed nosignificant differences between the responses of the two celllines. The effects of a range of doses of both drugs on therate of progress through the cell cycle was compared using cytofluorimetry.The more sensitive V79/79 cells failed to show a significantdelay in progress through the cell cycle even at the highestdoses tested (0.2 µM HN2 and 2.0 mM MMS). In contrast,V79 cells showed a marked S phase delay in response to bothHN2 and MMS exposure. The possible relationships between failureto delay cell cycle progression, and cellular sensivity arediscussed.     

Focus on cellular pharmacology of docetaxel

Docetaxel, a member of the taxoid class of antineoplasic agents, is derived from an inactive precursor obtained from the needles of the European yew. Docetaxel is characterised by a wide spectrum of in vitro cytotoxicity an murine and human tumour cell lines and in vivo antitumour activity against human tumour xenografted in athymic mice. The principal mechanism by which docetaxel exerts its cytotoxic activity is through enhancement and stabilisation of microtubule assembly leading to an arrest of the cell cycle at the G2/M phase. It has also been shown to induce tumour cell death by apoptosis and to have antiangiogenic and immunostimulating properties. Docetaxel demonstrated in vitro and/or in vivo additive and synergistic effects when combined with a large spectrum of anticancer therapies such as new cytotoxic agents (gemcitabine, capecitabine, edatrexate), specific signal transduction drugs, Herceptin, antiangiogenic drugs, gene therapy, or antisense olignonucleotides, opening to new treatment strategies. Despite close chemical structures, preclinical data suggested a difference in the cellular pharmacology of docetaxel and paclitaxel leading to different profiles in clinical studies.     

Antiproliferative response of human leukemic cells: lectin induced inhibition of DNA synthesis and cellular metabolism

Proliferation and DNA synthesis of human acute (CCRF-CEM, MOLT-4, JM, T-45) and chronic (SKW-3) lymphocytic leukemia cell lines of T-cell type were inhibited in a dose dependent fashion by the isolectins of phytohemagglutinin (PHA). PHA isolectin with four L subunits (PHA-L4) induced a significant antiproliferative response of CCRF-CEM and MOLT-4 tumor cells at a concentration of only 0.05 micrograms/ml. A 50% inhibition of DNA synthesis of these two cell lines was obtained at 0.4 and 0.5 micrograms PHA-L4/ml, respectively. The effect was cytostatic rather than cytotoxic. Considerably higher (greater than 10) concentrations of PHA isolectin with four E subunits were needed to induce similar growth inhibition of the tumor cells, as compared to PHA-L4. The effect of PHA isolectins on cellular metabolism of the leukemic cell lines was measured using microcalorimetry. The rate of heat production (thermal power), which is the net effect of all metabolic pathways, was decreased already after a 30-min culture of tumor cells in the presence of isolectin. PHA-L4 showed a significant inhibitory effect at a concentration of 0.1 micrograms/ml, whereas approximately 200 times more of PHA isolectin with four E subunits was needed to obtain the same metabolic inhibition. Measurements of glycolytic lactate and oxygen consumption during isolectin treatments supported the fact that the PHA-L4 induced antiproliferative response of human leukemic cell lines was specific and energy dependent. Six and three membrane components involved in the antiproliferative response of CCRF-CEM (ALL) and SKW-3 (CLL) cells, respectively, were isolated by affinity chromatography and characterized by electrophoresis.     

Dual mechanisms of inhibition of DNA synthesis by triciribine

The inhibition of DNA synthesis in triciribine (TCN)-treated L1210 cells was shown to involve two mechanisms, with different concentration dependence. (a) Initiation of new replicons and possibly of Okazaki fragments was inhibited when the cells were treated with 0.1 microM TCN. The inhibition of replicon initiation was shown by the rate of alkaline elution of [3H]DNA from 15-min-[3H]thymidine-labeled cells being slower if the cells had been pretreated with TCN, indicating that the average size of actively replicating DNA strands was increased. (b) At 1 microM TCN elongation of previously initiated DNA chains was also inhibited. This conclusion was suggested by the decrease in the rate of alkaline elution of [3H]DNA, during postlabeling incubation, being less if TCN was included in the medium. The mechanism of inhibition of DNA synthesis by TCN was shown not to involve DNA strand breakage or cross-linking, inhibition of polyamine biosynthesis, inhibition of purine de novo biosynthesis, inhibition of DNA polymerase alpha or DNA primase, or inhibition of ligation of Okazaki fragments. The effects of TCN on the incorporation of [3H]thymidine into Okazaki fragments and higher molecular weight DNA suggested the possibilities of inhibition of Okazaki fragment initiation and/or DNA polymerase delta.     

The apparent inhibition of urothelial DNA synthesis in neonatal rats by dietary saccharin

[³H]Thymidine ([³H]TdR) incorporation into urothelial DNA of male neonatal rats was measured autoradiographically at birth and during the first 3 weeks of life. The rats were derived from control parents and those fed saccharin (1, 3, 5 and 7.5%) in the diet from before pregnancy. [³H]TdR incorporation was inhibited and there were more lightly labeled cells (compared with controls), in all the saccharin-exposed rats in a rough dose-dependent manner. The results, in comparison with controls, suggest that saccharin exposure in utero causes DNA damage in the neonatal urothelium manifesting as reduced thymidine incorporation and a greater proportion of lightly labeled cells.     

The human cell multiprotein DNA replication complex (MRC): the effect of camptothecin on its ability to support in vitro DNA synthesis

 Purpose : We have previously reported on the isolation and characterization of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability to replicate DNA in a cell-free environment (devoid of other cellular processes) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor. Methods : We examined the effects of increasing concentrations of camptothecin on HeLa cell survival, intact HeLa cell DNA synthesis and MRC-mediated in vitro DNA replication. We also performed topoisomerase I assays in the presence of increasing concentrations of camptothecin to study the direct effects of the agent on MRC-associated topoisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleavable complexes in the presence of increasing concentrations of camptothecin. Results : We found a close correlation between the IC₅₀ values for intact HeLa cell DNA synthesis (0.15 μM) and MRC-mediated in vitro DNA synthesis (0.05 μM). Similarly, we found that 0.05 μM camptothecin inhibited MRC-associated topoisomerase I activity by approximately 50%. In addition, we found that the formation of MRC-associated topoisomerase I-cleavable complexes increased linearly with increasing concentrations of camptothecin. Conclusions: The data presented in this report support the use of the MRC as a model system to study the mechanism of action of camptothecin. We anticipate that future studies with the MRC will help elucidate the cellular consequences of camptothecin-cleavable complex formation. Received: 24 March 1995/Accepted: 22 March 1996     

Degradation of human exonuclease 1b upon DNA synthesis inhibition

In response to DNA damage, signaling pathways are triggered that either block the cell division cycle at defined transitions (G1-S and G2-M) or slow down progression through the S phase. Nucleases play important roles in DNA synthesis, recombination, repair, and apoptosis. In this study, we have examined the regulation of human exonuclease 1 (hEXO1b). The endogenous hEXO1b protein was only detected upon enrichment by immunoprecipitation. We found that hEXO1b was constantly expressed throughout the cell cycle. However, treatment of cells with agents that cause arrest of DNA replication led to rapid degradation of hEXO1b. This effect was fully reversed upon removal of the block. Analysis of synchronized cells showed that degradation of hEXO1b during the S phase was strictly dependent on DNA synthesis inhibition. DNA damage caused by UV-C radiation, ionizing radiation, cisplatin, or the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine did not affect hEXO1b stability. We show that hEXO1b was phosphorylated in response to inhibition of DNA synthesis and that phosphorylation coincided with rapid protein degradation through ubiquitin-proteasome pathways. Our data support the evidence that control of exonuclease 1 activity may be critical for the maintenance of stalled replication forks.     

Studies on cellular immunity and its serum mediated inhibition in Moloney-virus-induced mouse sarcomas

A colony-inhibition technique was used to demonstrate lymph-node cell (LNC) mediated immune reactions against tumor-specific transplantation antigens of primary, Moloney sarcoma virus induced mouse sarcomas. Lymph-node cells from mice in which Moloney sarcomas had regressed spontaneously (regressors), as well as LNC from mice carrying progressively growing tumors (progressors), induced by virus inoculation at an age of 14 days or older, reduced the plating efficiency of Moloney sarcoma target cells. Serum from mice with progressively growing Moloney sarcomas, but not from mice with spontaneous mammary carcinomas or methylcholanthrene-induced sarcomas, abrogated the inhibitory effect of regressor LNC on Moloney sarcoma cells. Additional evidence for the specificity of the serum effect was obtained in experiments showing that the protective effect of progressor sera could be specifically removed by absorption with Moloney sarcoma cells. Regressor sera gave no protection against target cell inhibition by regressor LNC. It is suggested that progressor sera contain antibodies which mediate an efferent form of immunological enhancement.     

Binding of daunomycin to DNA and the inhibition of RNA and DNA synthesis.

With synchronized tissue culture cells (L929), daunomycin had the greatest inhibitory effect on cell growth when the drug was administered during the later stages of cell division (late S, G2, and M). The level of binding of daunomycin to DNA was not found to be influenced by the phase of the cell cycle. The highest level of radioactivity from [eH]-daunomycin was bound to DNA of the heterochromatin fraction. Both RNA and DNA syntheses were inhibited in isolated enzyme systems when daunomycin-treated DNA, from which the unbound drug was removed by passage through Sephadex column, was used. DNA polymerase was reduced to one-fifth of the control activity, while that of RNA polymerase was reduced to one-half. Similar experiments with daunomycin-treated RNA and DNA polymerase preparations showed that the drug had no effect on the activities of the enzymes per se. Hence, the reduction of RNA and DNA polymerase activities could be accounted for by the loss of template activity of the drug-treated DNA. Daunomycin caused by a marked drop in the formation of a complex between RNA polymerase and DNA, indicating that the binding of daunomycin to DNA may give rise to steric hindrance effects that interfere with the association of the template to RNA polymerase enzyme. Sedimentation profile in alkaline sucrose density gradient of DNA that had been treated with daunomycin showed that no change in the molecular weight could be demonstrated.     

The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro.

The inhibition of 3H-thymidine incorporation into the DNA of mitogen-stimulated lymphocytes from patients with chronic lymphocytic leukaemia by chlorambucil was measured in vitro and the results related to clinical drug resistance. The assay proved to be both sensitive and specific showing a clear separation of those patients with responsive disease from those with disease resistant to treatment. There was evidence of primary drug resistance in untreated patients. In almost all patients who received treatment this led to increasing resistance to chlorambucil in vitro. The assay is predictive of clinical responsiveness and provides a potential means whereby new therapeutic agents and treatment modifiers may be investigated.     

Expression of SV40 T antigen during inhibition of DNA synthesis.

Treatment of human SV40-transformed cells with excess thymidine or cytosine arabinoside caused a rapid decrease in the expression of T antigen as determined by quantitative immunofluorimetry.