Brian W Fox

Differential cytotoxicity of Combretastatins A1 and A4 in two daunorubicin-resistant P388 cell lines

SummaryCombretastatin A4, a novel anti-mitotic agent was effective against two P388 cell lines with acquired resistance to daunorubicin. In contrast, Combretastatin A1, a close structural analogue of A4, showed a high degree of cross-resistance. Combretastatin A1 was also more efficient at increasing intracellular daunorubicin concentrations in both resistant cell lines. Neither agent was capable of altering anthracycline accumulation in the parental (sensitive) cell line. We propose that the cross-resistance to Combretastatin A1 occurs, at least in part, as a result of the increased affinity of the drug-efflux process operative in these resistant cells for Combretastatin A1 vs Combretastatin A4. Hence, Combretastatin A4 may play a role in the treatment of tumours with acquired resistance to the anthracycline antibiotics.

A proposed mechanism of resistance to cyclophosphamide and phosphoramide mustard in a Yoshida cell line in vitro

SummaryA Yoshida sarcoma cell line (YR/cyclo) showing decreased sensitivity to metabolically activated cyclophosphamide in vitro has been shown to be cross-resistant to phosphoramide mustard, the ultimate alkylating agent formed from cyclophosphamide. Resistance to these alkylating agents has been shown to be associated with increased activity of the glutathione S-transferase group of enzymes, and with elevated levels of glutathione, the cosubstrate of the enzyme. The resistant cell line shows lower levels of cellular damage, as measured by alkaline elution following treatment with phosphoramide mustard, than the parental (Ys) line. The mechanism of resistance is ascribed to increased deactivation of potentially damaging metabolites of cyclophosphamide by the glutathione S-transferase enzymes, resulting in decreased cellular damage in the resistant cell line.

The relationship between chromosomal aberrations, survival and DNA repair in tumour cell lines of differential sensitivity to X-rays and sulphur mustard

Abstract A pair of cultured rat lymphosarcoma cell lines (Yoshida) with a pronounced differential sensitivity to killing with sulphur mustard (SM), but with the same sensitivity to X-rays, was examined for chromosome damage and DNA repair replication after treatment with these agents. A pair of mouse lymphoma cell lines (L5178Y) with a differential sensitivity to X-rays was similarly investigated. SM-resistant Yoshida cells suffered much less chromosome damage than sensitive cells in spite of equal alkylation of DNA, RNA and protein in sensitive and resistant cells. The pair of Yoshida cell lines sustained the same amount of chromosome damage after X-irradiation. Much less chromosome damage was observed in the radiation-resistant lymphoma cell line than in the sensitive line after X-irradiation. No differences was found between the pairs of cell lines in their capacities for repair replication after SM or X-ray treatment. Thus, the drug and radiation resistance is accompanied by, and perhaps mediated through, a reduced amount of induced chromosome damage but is not quantitatively related to the capacity for DNA repair replication. Apart from small differences in modal chromosome numbers there are no obvious karyotype differences between the sulphur mustard-sensitive and -resistant Yoshida cells or between the radiation-sensitive and -resistant lymphoma cells.

The effect of ifosfamide and its metabolites on intracellular glutathione levels in vitro and in vivo.

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.

METALLOTHIONEIN LEVELS IN OVARIAN TUMOURS BEFORE AND AFTER CHEMOTHERAPY

The metallothionein content of ovarian tumours is considerably higher than that found in normal ovaries (greater than 100-fold differences in mean values, P less than 0.001). There was no difference between the metallothionein content of tumours from patients who had completed chemotherapy, usually with a regimen containing a platinum drug, and tumours from untreated patients. Similarly, the level of metallothionein was not influenced by response to therapy, age, stage, histology, or tumour cell differentiation state. These data do not support the hypothesis that metallothionein content is a major determinant of tumour sensitivity in ovarian cancer.

The role of metallothionein, glutathione, glutathione S-transferases and DNA repair in resistance to platinum drugs in a series of L1210 cell lines made resistant to anticancer platinum agents.

The glutathione contents, glutathione S-transferase activities and metallothionein contents have been measured in a series of L1210 cell lines which show decreased sensitivities to platinum drugs. Resistance to cisplatinum cisDDP, cis-diamminedichloroplatinum (II)] and chip [ioproplatin, cisdichloro-bis-isopropylamine-trans dihydroxy platinum IV] was found to correlate with glutathione levels but not metallothionein. Conversely, resistance to tetraplatin was found to be correlated with metallothionein but not glutathione levels. However, depletion of glutathione by buthionine 1-sulphoximine sensitizes all cell lines to the effects of cisDDP, chip and tetraplatin [d,1-trans-tetrachloro-1,2-diamino-cyclohexanplatinum (IV)]. Inhibition of DNA repair by aphidicholin or caffeine also partially restored sensitivity to these platinum drugs. These results indicate the complexity of the changes occurring upon the development of drug resistance.

DNA Single-strand Rejoining in Two Pairs of Cell-lines Showing the Same and Different Sensitivities to X-rays

SummaryThe numbers of single-strand breaks and their rates of rejoining have been compared in two Yoshida cell-lines showing similar sensitivity to X-rays (D0 85 rads, n = 2·5) and two L5178Y cell-lines showing differential sensitivity; the sensitive cell-line having a D0 value of 55 rads, n = 2·5, and the resistant line a D0 value of 160 rads and n = 3·0. After doses of 5 or 10 krads, no differences in the numbers of single-strand breaks produced or in their rates of rejoining were observed when measured by the alkaline sucrose gradient technique. The findings confirm that single-strand breaks in themselves are not lethal events, since similar numbers are produced and virtually all are rejoined in the cell-lines studied. However, a deficiency in some of the processes involved in repair replication could be the cause of their differential sensitivity by allowing damage to be fixed.

Bioactivity and molecular modelling of diphenylsulfides and diphenylselenides.

Bis(2-bromo-4,5-dimethoxyphenyl)sulfide (5) and bis(2-bromo-4,5-dimethoxyphenyl) selenide (7) have been shown to block cells in the G2/M phase of the cell cycle, whereas the debromo (4,6) equivalents do not. The biobromoselenide (7) is cytotoxic to tumour cells in vitro and has been shown to increase the mitotic index of treated cells. These biological effects are consistent with disruption of the mitotic apparatus. This agent does not inhibit microtubule assembly in vitro, but does bind to tubulin. Molecular modelling of these structures indicates that their spatial and electronic structures may make an important contribution to the biological activity.

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells.

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.

Sensitivity of the newly synthesized and template DNA of lymphoma cells to damage by methyl methanesulphonate, and the nature of associated “repair” processes

Abstract Using alkaline sucrose gradient centrifugation, single-strand breaks can be shown to occur in [ 14 C]thymidine labelled P388F lymphoma cells after treatment with methyl methanesulphonate at levels which allow for a high level of survival of the cells. The pattern of fragmentation of newly synthesized and template labelled DNA differs and these differences are discussed. The “repair” of this damage within 2-h after treatment has been studied and no evidence for rejoining of DNA strands during this time period was obtained. The increase in molecular weight following incubation of treated, newly labelled cells is now considered to be due to normal DNA synthesis on prelabelled fragments. The labelled template DNA shows two new features. A higher molecular weight component which appears to be broken internally by methyl methanesulphonate, but requires further incubation to release the fragments, and a characteristic distribution of labelled fragments which show no evidence of single-strand rejoining within 2-h post-treatment.