Regulation of growth of mouse mastocytoma cells.

N6,O2'-Dibutyryladenosine cyclic 3',5'-phosphate plus theophylline inhibited the growth of the mouse mast cell tumor line PY 815 both in vivo and in vitro. The inhibitory effect on growth in vitro was rapidly reversed following removal of the drugs. Growth inhibition was accompanied by reduced cell surface activity and increased cell-cell adhesion. The drug-treated cells accumulated distinct membrane-bound granules, which are characteristic of more mature mast cells. Treated cells also developed increased amounts of surface-associated acidic mucopolysaccharides. These results suggest that increased intracellular cyclic adenosine 3':5'-monophosphate causes mouse mastocytoma cells to decrease growth and elicits the expression of a more differentiated mast cell phenotype. The effect of the antileukemia drug, 4'-(9-acridinylamino)methanesulfon-m-anisidine, on cyclic adenosine 3':5'-monophosphate and adenosine 5'-triphosphate in mastocytoma cells is also reported.

Cyclic-AMP-induced c-fos expression and its relevance to differentiation of a transformed mast cell line.

The effects of cyclic AMP on expression of the oncogenes c-myc, c-myb and c-fos in murine P815 mastocytoma cells were examined in relation to growth and differentiation. Induction of differentiation in mastocytoma cells by cyclic AMP was accompanied by a rapid increase in c-fos expression. Cyclic AMP induced stable expression of c-fos mRNA by increasing c-fos transcription 4-5-fold and slightly increasing the stability of c-fos mRNA. However, a high level of c-fos expression was not essential for differentiation of two temperature sensitive-mutant P815 cell lines, as c-fos mRNA did not increase in differentiating temperature-sensitive P815 cells. These results do not support an essential role for c-fos expression in the differentiation of mast cells. Although c-myc expression was lower after growth arrest by cyclic AMP, this decrease did not correlate with growth inhibition by cyclic AMP, since c-myc expression decreased only after cells had started to arrest in G1 phase.

A protein factor that enhances amsacrine-mediated formation of topoisomerase II-DNA complexes in murine mastocytoma cell nuclei.

Extracts of K21 murine mastocytoma cells contain a factor that enhances formation of amsacrine-induced topoisomerase II-DNA complexes (PDCs) when added to isolated K21 nuclei. The PDC-enhancing activity is reduced in extracts from 2 or 6 h cycloheximide or cordycepin-treated cells, implying that continuous protein synthesis is required to maintain the factor. The factor is heat-labile, proteinase-sensitive and has other properties that distinguish it from the two known classes of topoisomerases. The data suggest that the factor is a labile protein with a molecular weight in excess of 50,000. This appears to be the first direct evidence of a protein factor that modulates drug-induced topoisomerase II action.

Regulation of topoisomerase II by murine mastocytoma cells.

Nuclei from K21 murine mastocytoma cells do not form topoisomerase II-DNA adducts in response to amsacrine in the absence of a cytoplasmic factor tentatively identified as a type of casein kinase (Darkin, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291). The stimulatory activity was present in extracts from cells grown in horse serum but not in calf serum. Activity was lost following growth arrest by serum deprivation. In contrast, topoisomerase II activity in isolated nuclei did not decline during growth arrest. These results suggest that the resistance of some non-cycling tumour cells to anti-cancer drugs may result from decreased activation of topoisomerase II.

Inhibition of turnip yellow mosaic virus synthesis by pyrimidine analogues.

The pyrimidine analogues 2-thiouracil, 2-thiouridine, 6-azauracil and 6-azauridine all inhibited the synthesis of turnip yellow mosaic virus (TYMV) and increased the synthesis of empty virus protein shells in infected Chinese cabbage leaf discs. Uracil and uridine reversed these effects. 2-Thiouracil also reduced the UTP pool in TYMV infected leaf discs. The results are consistent with the suggestion that these analogues or their in vivo derivatives affect virus synthesis by inhibiting the biosynthesis of uridylic acid, possibly by inhibiting orotidylic acid decarboxylase.

Cyclic AMP calcium and the growth of mastocytoma cells.

Arresting P815 mastocytoma cell growth with N6, O2'-dibutyryladenosine 3':5' cyclic monophosphate (db cAMP) and theophylline increased 45Ca2+ uptake and efflux by the cells (i.e, Ca2+ cycling) without altering cytoplasmic free Ca2+ concentrations or the amount or distribution of protein kinase C in the cells. Attempts to identify the Ca2+ channels involved using a wide variety of drugs were unsuccessful. However, the inhibitory effect of db cAMP on growth was greatly increase in medium containing low Ca2+ concentrations, confirming that interactions between Ca2+ and cyclic AMP can affect mastocytoma cell growth.

Evidence that a protein kinase enhances amsacrine mediated formation of topoisomerase II-DNA complexes in murine mastocytoma cell nuclei.

Cytoplasmic extracts of K21 murine mastocytoma cells contain a protein factor, distinct from topoisomerases I and II, that facilitates formation of amsacrine-induced topoisomerase II-DNA complexes (PDC) in isolated K21 cell nuclei (Darkin, S.J. and Ralph, R.K. (1988) Biochim. Biophys. Acta 1007, 295-300). The PDC enhancing activity was shown to reside in a protein kinase with specificity for a casein kinase II substrate and sensitive to heparin and anti-casein kinase II antiserum. This appears to be the first direct evidence of a protein factor that modulates amsacrine-induced topoisomerase II action.

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.

Mechanism of resistance of non-cycling mammalian cells to 4'-[9-acridinylamino]methanesulphon-m-anisidide: role of DNA topoisomerase II in log- and plateau-phase CHO cells.

CHO-AA8 cells were used as a model system to study the role of DNA topoisomerase II in the resistance of non-cycling cells to amsacrine. Plateau-phase AA8 cells have previously been shown to be resistant to amsacrine and to contain fewer DNA breaks than log-phase cells after drug treatment (Robbie, M.A., Baguley, B.C., Denny, W.A., Gavin, J.R. and Wilson, W.R. (1988) Cancer Res., in press). The phage P4-unknotting activity of nuclear extracts decreased 2-fold when AA8 cells entered into the non-cycling state, but there was no difference in sensitivity to amsacrine between log- and plateau-phase nuclear extracts. Drug stimulation of protein-DNA complex formation was similar in whole cells, isolated nuclei and nuclear extracts from either log- or plateau-phase cells. However, stimulation of complex formation in cells, nuclei or nuclear extracts was approx. 4-fold lower in plateau-phase than in log-phase. The data presented suggested that drug-enzyme interaction was altered in plateau-phase cells.

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line.

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.

Cyclic AMP and Ca2+ uptake by mastocytoma mitochondria.

A thorough re-investigation was undertaken of a variety of factors that might explain the increased uptake of 45Ca2+ by mitochondria isolated from N6, O2'-dibutyryladenosine-3',5'-cyclic monophosphate (DB cyclic AMP)--treated PY815 cells. This showed that mitochondria isolated from DB cyclic AMP treated cells take up 45Ca2+ at a 30 per cent faster rate than mitochondria from untreated cells, although both mitochondria eventually reduce the total external Ca2+ to the same levels. 45Ca2+ precharged mitochondria from DB cyclic AMP-treated cells also leaked 45Ca2+ more slowly than those from untreated cells when they were recovered by filtration. Thus an apparently greater uptake of 45Ca2+ by mitochondria from DB cyclic AMP-treated cells was a consequence of the filtration procedure. In fact, mitochondria from DB cyclic AMP-treated cells contained less total Ca2+ than those from untreated cells, while DB cyclic AMP-treated cells also contained less total Ca2+ than untreated cells. The results suggest that mitochondria do not play an important role in controlling the growth of DB cyclic AMP-treated PY815 cells through effects on cytoplasmic Ca2+ availability.

Dibutyryl cyclic AMP effects on calcium metabolism by mouse mastocytoma cells.

The ability of mouse mastocytoma cells to take up 45Ca2+ was measured in normal growth medium. As previously observed in physiological buffers with succinate and Pi, cells grown for 18h with N6,O2'-dibutyryladenosine 3',5' cyclic monophosphate (DB cyclic AMP) to inhibit growth took up more 45Ca2+ than untreated cells. However 45Ca2+ uptake by cells in growth medium was less sensitive to respiratory inhibitors or uncouplers than 45Ca2+ uptake in physiological buffer. Increased 45Ca2+ uptake by 18h cyclic nucleotide-treated cells was not a result of tighter mitochondrial coupling since mitochondria prepared from cyclic nucleotide-treated cells were less coupled than those from untreated cells. Nevertheless studies with uncouplers suggested that the bulk of the intracellular Ca2+ was associated with mitochondria. DB cyclic AMP-treated cells contained less total Ca2+ than untreated cells indicating that net Ca2+ efflux occurred during the 18h period of drug treatment. These observations suggest that Ca2+ fluxes increase in DB cyclic AMP-treated PY815 cells and that a net efflux of Ca2+ occurs during growth inhibition by the cyclic nucleotide.

Cyclic AMP, calcium and control of cell growth.

The role of cyclic AMP and calcium in the control of normal and tumour cell growth is considered in relation to the question whether cyclic AMP is a true mitogen or co-mitogen. It is proposed that cyclic AMP normally controls the cell cycle at a point in G1 phase only by virtue of its ability to exclude calcium required by cells to progress past this point into S phase. Therefore increased influx of calcium by other routes induced by various factors can bypass the inhibitory effect of cyclic AMP and stimulate growth. In these circumstances cyclic AMP or calcium may or may not facilitate further progress into S phase according to the metabolic requirements of individual cells. The relevance to cancer cells is considered.

Transport of AMSA drugs into cells.

The uptake and efflux of radioactive 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA) and its inactive congener 4'-(9-acridinylamino)methanesulphon-o-anisidide (oAMSA) by PY815 mastocytoma cells were investigated. Both drugs were readily taken up by intact cells although only mAMSA caused DNA scission and is actively cytotoxic to PY815 cells. The microsomal enzyme inhibitors cimetidine or SKF525A increased drug uptake and decreased drug efflux suggesting that drug metabolism could explain the different activities of oAMSA and mAMSA.

Evidence that mAMSA induces topoisomerase action.

Evidence is presented that the topoisomerase inhibitors novobiocin and coumermycin inhibit the production of double-strand breaks in mouse mastocytoma cell nuclear DNA by the anticancer drug 4'[(9-acridinyl)amino]-methanesulphon-m-anisidide (mAMSA). Novobiocin did not inhibit resealing of DNA breaks induced by mAMSA. It is suggested that mAMSA intercalation into DNA induces the action of a type II topoisomerase. mAMSA and oAMSA were equally effective in breaking the DNA in isolated nuclei.

Cyclic AMP and c-myc gene expression in PY815 mouse mastocytoma cells.

The possibility was examined that inhibition of growth of PY815 mouse mastocytoma cells by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (DB cyclic AMP) results from inhibition of c-myc gene expression. Temporary increases in c-myc RNA which occurred soon after DB cyclic AMP treatment and upon removal of the drug were not consistent with direct inhibition of c-myc gene expression by DB cyclic AMP. The increases in c-myc RNA coincided with the passage through, or accumulation of cells in late G1-early S phase. It is proposed that cyclic AMP may stimulate c-myc gene expression which normally occurs only in late G1-early S phase in PY815 cells and that cyclic AMP prevents c-myc expression in cells at other phases of the cell cycle by inhibiting their progression past a cyclic AMP-sensitive restriction point in early G1 phase.

Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum.

A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions. 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices. A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods. Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position. In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM). Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme. The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM. It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above. In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM. Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay. The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P. falciparum in vitro (IC50 20 nM) and P. falciparum-derived topoisomerase II. However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.

Structure-activity relationships for the antileishmanial and antitrypanosomal activities of 1'-substituted 9-anilinoacridines.

Members of the class of 9-anilinoacridine topoisomerase II inhibitors bearing lipophilic electron-donating 1'-anilino substituents are active against both the promastigote and amastigote forms of the parasite Leishmania major. A series of analogues of the known 1'-NHhexyl lead compound were prepared and evaluated against L. major in macrophage culture to further develop structure-activity relationships (SAR). Toxicity toward mammalian cells was measured in a human leukemia cell line, and the ratio of the two IC50 values (IC50(J)/IC50(L)) was used as a measure of the in vitro therapeutic index (IVTI). A 3,6-diNMe2 substitution pattern on the acridine greatly increased toxicity to L. major without altering mammalian toxicity, increasing IVTIs over that of the lead compound. The 2-OMe, 6-Cl acridine substitution pattern used in the antimalarial drug mepacrine also resulted in potent antileishmanial activity and high IVTIs. Earlier suggestions of the utility of 2'-OR groups in lowering mammalian cytotoxicity were not borne out in this wider study. A series of very lipophilic 1'-NRR (symmetric dialkylamino)-substituted analogues showed relatively high antileishmanial potency, but no clear trend was apparent across the series, and none were superior to the 1'-NH(CH2)5Me subclass. Subsets of the most active 1'-N(R)(CH2)5Me- and 1'-N(alkyl)2-substituted compounds against L. major were also evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei, but no consistent SAR could be discerned in these physiologically diverse test systems. The present study has confirmed earlier conclusions that lipophilic electron-donating groups at the 1'-position of 9-anilinoacridines provide high activity against L. major, but the SAR patterns observed do not carry over to the other parasites studied.

Decreased inhibition by gravidin of arachidonate release from transformed compared to nontransformed cells.

1. Gravidin (a phospholipase A2 inhibitor) reduced the release of arachidonic acid from human lymphocytes by 51% at 10(-8) M. 2. Under normal culture conditions, nanomolar gravidin caused a significant reduction in the release of free arachidonic acid from human lymphocytes or nontransformed fibroblasts but in transformed cells, nanomolar gravidin was ineffective. 3. Inhibition of arachidonate release appeared to be related to rate of growth as inhibitory effects of gravidin on Jurkat cells and HL-29 cells could be observed if the cells were cultured under conditions where DNA synthesis was low. 4. The reported disparate effects of lipocortin on cell phospholipase A2 activity may be reconciled if DNA synthesis is investigated.

Potentiation of 4'-(9-acridinylamino)methanesulphon-m-anisidine) action by verapamil.

Verapamil was shown to increase growth inhibition and decrease viability of PY815 mastocytoma cells treated with the anti-cancer drug mAMSA 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA) or its normally inactive congener, 4'-(9-acridinylamino)methanesulphon-o-anisidide (oAMSA). Verapamil also potentiated the effect of sub-optimal concentrations of mAMSA or oAMSA on DNA scission in intact cells. Uptake of [14C]mAMSA by PY815 cells was considerably enhanced, while efflux of [14C]mAMSA from precharged cells was inhibited by verapamil. It is concluded that verapamil potentiates the action of mAMSA on PY815 cells in culture by reducing efflux of drug from the cells. The possibility that verapamil may affect systems that sequester or metabolize AMSA drugs is suggested.