Inhibitory effects of 9-anilinoacridines on Plasmodium falciparum gametocytes.

Two gametocyte-producing isolates of Plasmodium falciparum, KT1 arid KT3, were cultivated in vitro. On day 11 of cultivation, pure gametocytes containing stage II, III and IV were used to test the gametocytocidal activity of 9-anilinoacridines that had previously demonstrated their activity against the asexual stage of the parasite. After drug exposure for 48 h, gametocytes were maintained without drugs for another 2 days before thin films were prepared for parasite counting. Gametocytocidal activities of 13 analogs of 9-anilinoacridine were observed with 50% inhibitory concentrations in the range of 0.6 microM to> 100 microM The most active compound was 1'-CH2NMe2-9-anilinoacridine. Anilinoacridine derivatives with 3,6-diamino substitution had reduced gametocytocidal activity in contrast to their enhancing effect against the asexual forms. Morphological abnormalities of gametocytes were observed following drug exposure.

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.

Structure-activity relationships of 9-anilinoacridines as inhibitors of human DNA topoisomerase II.

A series of 9-anilinoacridines, based on the anticancer drug amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide; m-AMSA], were synthesized and evaluated for their ability to inhibit both the growth of Jurkat leukaemia cells and human DNA topoisomerase II in vitro. Inhibition of topoisomerase II activity occurred via one of two mechanisms of drug action: (i) direct inhibition of the strand-passing activity or (ii) stabilization of cleavable complex formation. Although the majority of compounds evaluated inhibited P4 DNA unknotting activity catalysed by DNA topoisomerase II up to 100 microM, derivatives bearing 1'-substituents containing SO2 moieties (e.g. 1'-NHSO2Me and 1'-SO2NH2 groups) were generally the most potent inhibitors of DNA topoisomerase II-mediated DNA religation, being effective at concentrations of 1-5 microM. No obvious correlation was observed between the cytotoxicity of individual drugs and linear DNA formation in the in vitro topoisomerase II assays, either across the whole drug series or within similar subgroups. However, a selected group of drugs with different cytotoxicities (compounds 5, 12 and 30; Table I) stimulated DNA topoisomerase II-mediated DNA strand breaks in intact Jurkat cells by 3.5-, 11- and 2.2-fold, respectively, at a concentration of 10 microM, while compounds 31 and 32 did not produce protein-associated DNA strand breaks in whole cells. There was a good correlation between the ability of these selected compounds to induce topoisomerase II-mediated DNA strand breaks in vitro or in whole cells, and their cytotoxicity to Jurkat cells.

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.

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.

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.

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.

Growth-related protein kinases.

A protein kinase cascade is involved in the action of some mitogens. The cascade begins with receptor tyrosine kinase activation by growth factors. The resulting signal is transmitted into cells via phospholipid metabolism which produces a variety of second messengers and by intracellular protein kinase activation. The signal is then propagated and disseminated via a network of other protein kinases and protein phosphatases. Recent research suggests that ribosomal protein S6 kinase and casein kinase II are two important elements in the kinase cascade that leads to the initiation of growth. The nature and some properties of these hitherto lesser known enzymes is considered.

Inhibitors of lipoxygenase have antiproliferative effects on P815 murine mastocytoma cells.

The effect of a variety of inhibitors of prostaglandin synthase, lipoxygenase and phospholipase on the growth of P815 murine mastocytoma cells was examined. Only lipoxygenase inhibitors substantially reduced growth, presumably by inhibiting the production of arachidonic acid metabolites rather than causing arachidonate accumulation since excess arachidonic acid did not reverse growth inhibition. Evidence is presented that production of leukotrienes B4, C4, D4 or E4 was not involved. Other metabolites of arachidonic acid were not excluded. A role for lipoxygenase in growth signal transduction in these and other cells is suggested.

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.

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.

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.

Inhibition of protein synthesis reduces the cytotoxicity of 4'-(9-acridinylamino)methanesulfon-m-anisidide without affecting DNA breakage and DNA topoisomerase II in a murine mastocytoma cell line.

Stimulation of cleavable complex formation by 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) and related anticancer drugs is an important initial event in drug action which correlates with cytotoxicity. However, it was recently suggested that factors in addition to cleavable complex formation are needed to express lethality. Therefore we investigated the effects of inhibitors of DNA replication and RNA and protein synthesis on mAMSA-induced cell killing in the K21 subline of the P815 murine mastocytoma cell line. This showed that RNA and protein synthesis, but not DNA replication, was necessary for maximal mAMSA cytotoxicity. Moreover, inhibition of RNA synthesis with cordycepin or protein synthesis with cycloheximide protected cells from the cytotoxic action of mAMSA without reducing DNA breakage or cleavable complex formation and there was no decrease in DNA topoisomerase II activity in nuclear extracts from cells treated with cordycepin or cycloheximide. We conclude that cleavable complex formation is independent of RNA and/or protein synthesis and we propose that the subsequent conversion into a lethal event requires an additional labile protein factor.

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.

Cell line selectivity and DNA breakage properties of the antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide: role of DNA topoisomerase II.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (NSC 601316) is a DNA intercalating experimental antitumour agent which is curative against the Lewis lung carcinoma in mice. Its action has been compared with amsacrine, its inactive isomer oAMSA, the solid tumour active derivative CI-921 (NSC 343499), a C-6 methylene chain-linked bisacridine (NSC 210733), 9-aminoacridine and quinacrine. All compounds inhibited the unknotting of phage P4 DNA by topoisomerase II in nuclear extracts prepared from L1210 cells. NSC 601316 inhibited growth of cultured L1210, P388, P/AMSA (P388 resistant to amsacrine) and P/ACTD (resistant to actinomycin D) cell lines at concentrations of 87, 150, 2020 and 150 nM respectively. A 1 h drug exposure to 0.85 microM NSC 601316 killed 50% of L1210 cells. L1210 cells treated for 1 h with NSC 601316 accumulated DNA breaks and protein-DNA cross-links. There was a good correlation between DNA breakage and cytotoxicity, but the relationship between drug concentration and number of protein-DNA cross-links was non-linear and differed from that of amsacrine and CI-921. There was also a positive correlation between the degree of cross-resistance of P/AMSA cells (which have altered topoisomerase II function) and ability to induce DNA breakage or protein-DNA complexes. The results suggest that topoisomerase II is the target of action of NSC 601316.

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.

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.

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.

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.