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Biomedical subjects

R K Ralph

Publications and source records attributed to R K Ralph.

At least 19 recordsLinked to original sources
PubMed1992

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.

Amsacrine↗
PubMed1992

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.

Arachidonic Acid↗
PubMed1991

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.

Amsacrine↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Amsacrine↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Amsacrine↗
PubMed1988

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.

Amsacrine↗
PubMed1988

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.

Amsacrine↗
PubMed1988

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.

Acridines↗
PubMed1986

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.

Adenosine Triphosphate↗
PubMed1986

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.

Aminoacridines↗
PubMed1985

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.

Aminoacridines↗
PubMed1985

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.

Animals↗
PubMed1985

Cyclic AMP, nuclear protein kinase and the PY815 cell cycle.

A substantial increase in cyclic AMP-dependent protein kinase activity occurred in nuclei of PY815 mastocytoma cells during G1 phase growth arrest by DB cyclic AMP and the increased nuclear protein kinase was accompanied by changes in nuclear protein phosphorylation. However, there was no obligatory association between the rise in nuclear cyclic AMP-dependent protein kinase in G1 phase and growth arrest because nuclear cyclic AMP-dependent protein kinase also increased during G1 phase in cycling PY815 cells synchronized with amethopterin. These observations suggest that maintenance of high cyclic AMP levels during G1 phase may cause growth arrest by activating a cyclic AMP-dependent protein kinase that normally increases in PY815 cell nuclei during G1 phase.

Animals↗