Cytotoxicity Of Mitomycin Research Articles

In vitro evaluation of cytotoxic properties of 5-Aminolevulinic acid (5-ALA) on bladder cancer cells.

5-Aminolevulinic (5-ALA) may be used as a photo diagnostic agent in bladder cancer. The aim of this study was to investigate the cytotoxic effect of 5-ALA on bladder cancer cells. T24 cells treated with various concentrations of mitomycin (MC), 5-ALA and an MC/5-ALA mixture were evaluated to determine the role of 5-ALA on MC cytotoxicity. Cell cycle analysis was conducted, and apoptosis was analyzed by flow cytometry. Caspase 3 enzyme and reactive oxygen species were measured. Our initial studies exploring the impact of combination therapy on cell viability demonstrated improved cytotoxic effects on T24 and RT cells with relatively low doses of 5-ALA/MC in conjunction with MC alone. Indicated no significant difference between the IC50 of MC and MC/5-ALA in T24 cells, while IC50 value was decreased by 25 % in RT4 cells in 5-ALA/MC in comparing with MC alone. However, examination of cell cycle phase arrests by flow cytometry revealed significant PreG1 apoptosis and cell growth arrest in G2/M in T24 cells treated with the MC/5-ALA mixture compared with MC treatment. In addition, caspase 3 enzyme was increased by 1.15-fold in T24 cells treated with MC/5-ALA in comparison with MC alone. These results suggest that 5-ALA might possess anti-cancer properties and is not only a photo diagnostic element.

First multicenter experience with thermotherapy and mitomycin of high risk NMIBC with a new recirculation system (COMBAT).

425 Background: At a temperature of 43° (109°F) cytotoxicity of mitomycin increases 10 fold to tumor cells if used for instillation therapy of superficial bladder cancer (NMIBC).In the last years high organ preservation rates were reported for high risk NMIBC after radiofrequency induced hyperthermia and mitomycin (Synergo System).Unfortunately side effects were frequent and discontinuitation of 40% were reported with this method thus thermotherapy combined with instillation this therapy has not been very common.We here report about the first experience with a newly developed device using a recirculation heating system (COMBAT). Methods: After diagnosis by complete TUR/ReTUR patients received 40 mg mitomycin (mito-extra,medac) via transurethral catheter for 1 h heated up to constant 43° by a circulation pump system (COMBAT). Side effects were recorded pprospectively before and after each teratment and during follow up and success was validated by TUR/biopsy if possible. Results: A total of 20 patients were treated from 11/14-9/15 in 5 institutions. Follow up until now is 1-10 month (mean 8 m). All patients had high risk NMIBC including BCG failures. Therapy must be abondened in 1 patient after the 5th treatment, 50% had dysuria during treatment but symptoms decreased after the last treatment in all patients. 2 patients experienced mild allergic reaction also seen under conventional instillation therapy with mitomycin and were treated by corticosteroids. ReTUR was performed in 17/20 patients and revealed a persistent/recurrent tumor pTa GIII in 1 out of 20 patients. Conclusions: Compared to the microwave driven heating system the new device with recirculation of heated mitomycin seems to have less side effects without major complications. Treatment discontinuitation was recorded in 1/20 patients. Given the assumption that this system has the same high rate of organ preservation as the Synergo System at even much lower costs, it may alter the therapy algorythm in high risk NMIBC. Despite our first promising oncological results further research with longer follow up is necessary.Two prospective randomized studies are now underway in England and Spain and first results are awaited in 2 years.

Reductive activation of mitomycins A and C by vitamin C

The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.

Optimization of fluorescent cell-based assays for high-throughput analysis using microchamber array chip formats

This study describes the optimization of fluorescent cell-based assays using microchamber array chip formats as well as using automatic nanoliter volumes of sample dispensing system for high-throughput screening analysis of anticancer drugs. Cell-based assays can be employed efficiently in the screening of potential anticancer drug candidates and bioactive compounds with distinct biological function. Identification and development of cell-based assays adapted to high-throughput screening requirements is important when screening chemicals for their potential anticancer properties. Cell-based screening assays using microchamber array chip formats and automatic nanoliter volumes of sample dispensing system requires an optimization as a prerequisite for parameters including assay liquid volume and number of cells per each chamber, and the total cell-based assay itself. Further, the anticancer effect of mitomycin C was studied as an example against human cervical carcinoma cell line-HeLa 229 using cell-based assay that was optimized on chamber array chip formats and determined the cytotoxicity of mitomycin C by measuring the cell proliferation of HeLa with Calcein-AM fluorescent dye. The cell-based screening assay that was performed using chamber array chip formats was compared with the conventional 96-well plate formats was discussed. The assay described in this study is rapid, simple and inexpensive that is desirable in selecting anticancer candidates.

Cellular resistance to mitomycin C is associated with overexpression of MDR-1 in a urothelial cancer cell line (MGH-U1).

To compare multidrug resistance (MDR)-1 and MDR-3 gene expression in a new urothelial cancer cell line (MGHU-1, with resistance to mitomycin C) against controls and the established (epirubicin-resistant) MDR clone, and to correlate MDR with cytotoxicity data. Resistance to mitomycin C was induced by the long-term exposure of wild-type MGHU-1 cells to increasing concentrations (20-400 nmol/L) of mitomycin C. The cytotoxicity of mitomycin C or epirubicin was then compared in MGHU-1, MGHU-MMC (mitomycin C-resistant) and MGHU-1R (established MDR) cells, using the tetrazolium biomass assay. The expression of MDR-1 and -3 was investigated by the reverse transcriptase-polymerase chain reaction, using cDNA-specific primers after titration, and compared with DNA and negative controls. MDR-1 and -3 were significantly and equally overexpressed in MGHU-1R, and associated with a dramatic increase in the 50% inhibitory drug concentration (P < 0.001) for mitomycin C and epirubicin against controls. In MGHU-MMC, the overexpression of MDR-1 was three times greater than that of MDR-3. The cytotoxicity profile for both agents was very similar to that of MGHU-1R. Trace amounts of MDR-1, but not MDR-3, were identified in the MGHU-1 wild-type. Urothelial cancer cell resistance to mitomycin C is associated with cross-resistance to epirubicin and overexpression of MDR-1, suggesting that mitomycin C falls within the MDR category. Clinical application of this methodology may allow patients to be identified who are unlikely to benefit from intravesical chemotherapy.

The Suppressing Effect of the Extract from Cassia nomame on Clastogenicity and Cytotoxicity of Mitomycin C in CHO Cells.

Chinese hamster ovary (CHO) K1 cells were treated with 2.5 μM Mitomycin C (MMC) for one hour and incubated in Ham’s F12 medium containing 10% fetal bovine serum or in the medium supplemented with the extract from Cassia nomame pods, leaves and stems. The frequency of cells with chromosome aberrations was significantly lowered by the extract. Proliferation of CHO cells was suppressed by a 1-hr MMC treatment, but the suppression was nullified by the presence of the extract. These findings suggest that the extract can prevent the physiological damage caused by MMC.

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postla-beling and was characterized as a monoadduct ofguanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations atG:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations. Environ. Mol. Mutagen. 29:143–151, 1997. © 1997 Wiley-Liss, Inc.

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

Sensitivity of cell lines to mitomycin C

To determine the effect of pH on the cytotoxicity of mitomycin C on a wide range of established cell lines. Cultures of three human tumour cell lines, including two bladder carcinomas and one renal adenocarcinoma, and two mouse cell lines were treated with mitomycin C (concentration range 320 pM-50 microM) at a range of pH (5.8-7.8) for 2 h. The cytotoxicity of mitomycin C over this range was determined using a tetrazolium-dye-based microtitration assay. The cytotoxicity of mitomycin C was significantly greater (confidence level > or = 95%) in acidic conditions (pH 5.8-6.0) than in neutral to alkaline pH (7.2-7.8) for three of the five cell lines. The same trend, although less marked, also occurred in the other two cell lines. The cytotoxicity of mitomycin C was greater under acidic conditions for all these cell lines confirming that the activity of mitomycin C depends on pH and suggesting that the pH of the vehicle used for intravesical chemotherapy may be an important factor in the successful treatment of bladder carcinomas. The modification of pH by urine may explain some of the variation in success rates among patients treated intravesically with mitomycin C.

The cytotoxicity of mitomycin C and Adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and β-naphthoflavone (βNF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and α-naphthoflavone (αNF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by aNF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or βSNF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in βNF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas aNF only partially inhibited the cytotoxicity of MMC in βNF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.

Synergistic anti-tumor effects of mitomycin C and bile salts against L1210 cells.

The effects of various adjuvants on the cytotoxicity of mitomycin C (MMC) were studied in L1210 mouse leukemia cells. Adjuvants examined in this study were sodium glycocholate (Na-GC), sodium deoxycholate (Na-DC), O-n-dodecyl-beta-D-maltopyranoside (LM), and sodium salicylate. Among various additives, bile salts such as Na-GC and Na-DC were the most effective for increasing the cytotoxicity of MMC against L1210 cells. A dose-dependent increase in cytotoxic effect of MMC was observed in the presence of these bile salts. To elucidate a possible mechanism for enhancing the cytotoxic effect of MMC by the bile salts, the cellular uptake of MMC with or without Na-GC was examined using L1210 cells. The cellular concentration of MMC was determined by a reversed-phase HPLC. When Na-GC was coadministered with MMC, the uptake of MMC into L1210 cells was significantly enhanced as compared with MMC alone. Furthermore, the membrane fluidity of L1210 cells, as determined by fluorescence polarization, was increased in the presence of Na-GC. These results suggested that the enhancement of cytotoxicity of MMC by the addition of Na-GC could be attributed to the increasing cellular uptake of MMC due to the increasing membrane fluidity of L1210 cells.

Minocycline as a modulator of chemotherapy and hyperthermia in vitro and in vivo

We tested the ability of the collagenase-inhibitor minocycline to increase the effectiveness of CDDP, BCNU and mitomycin C ± hyperthermia. When tested in vitro in FSaIIC fibrosarcoma cells, exposure to minocycline (100 μM for 24 h) decreased the CDDP cytotoxicity at 37°C and pH 7.40 in both normally oxygenated and hypoxic cells and decreased the cytotoxicity of CDDP at 42°C or 43°C in normally oxygenated cells while increasing the killing in hypoxic cells. When tested at pH 6.45, the presence of minocycline tended to protect both normally oxygenated and hypoxic cells from the cytotoxic effects of CDDP ± hyperthermia. With exposure to BCNU, minocycline markedly protected both normally oxygenated and hypoxic cells at 37°C at both pHs. As the temperature during the exposure to BCNU was increased to 42°C or 43°C, the protection afforded by minocycline diminished especially under low pH conditions where BCNU plus 43°C was extremely cytotoxic to both normally oxygenated and hypoxic cells. One hour exposure to mitomycin C was more cytotoxic to hypoxic than normally oxygenated cells under all conditions of pH and temperature tested and the cytotoxicity of mitomycin C under each condition was increased by minocycline. Both CDDP and BCNU were much more cytotoxic toward FSaIIC tumors in vivo when drug administration was followed by local heating (43°C, 30 min) of the tumor bearing limb. In each case, treatment with minocycline had little effect on tumor-cell killing. Treatment with mitomycin C and hyperthermia resulted in additive tumor-cell killing, and minocycline administration further increased that effect. Tumor-growth delay studies in FSaIIC tumor-bearing animals demonstrated that treatment with CDDP, BCNU or mitomycin C produced growth delays which were additive with hyperthermia (43°C, 30 min) and/or minocycline administered daily for 14 days on days 4–18. These results indicate that minocycline, has measurable antitumor and cytotoxic effects under the conditions tested.

The effect of low ph and hypoxia on the cytotoxic effects of SR4233 and mitomycin C in vitro

Purpose : We examined the effect of acidic pH and hypoxia on the cytotoxicity of SR4233 and mitomycin C in vitro. Methods and Materials : The importance of tumor microenvironment to the response of solid tumors to cytotoxic treatment is well established. The bioreductive drug SR4233 has a very substantial selective toxicity for hypoxic cells. We have used both Chinese hamster and human tumor cells to investigate the influence of low pH and hypoxia on the response of cultured cells to treatment with SR4233 or mitomycin C. Results : We found that low pH (6.6) had little effect on the hypoxic toxicity of SR4233; under aerobic conditions, however, low pH substantially increased the cytotoxic effects of 1 h exposure to SR4233, with drug dose enhancement ratios (ER) of 3.9 and 2.5 in V79 and HT-29 cells, respectively. In similar studies with mitomycin C, hypoxia had little effect on the cytotoxicity of mitomycin C in V79 cells, though a low pH of 6.6 enhanced the cytotoxicity under both aerobic and hypoxic conditions (ER ∼ 2). In HT-29 cells, neither low pH nor hypoxia had any significant effect on mitomycin C toxicity. Conclusion : Low pH, like hypoxia, is a common feature of solid tumors and can be an important determinant of the cytotoxic effect of bioredutive drugs such as SR4233 and mitomycin C.

Potentiation of cytotoxicity of mitomycin C by a polyacetylenic alcohol, panaxytriol.

Polyacetylenic alcohol, panaxytriol, which was isolated from Panax ginseng C. A. Meyer, has antiproliferative activity against several kinds of tumor cells. In this paper, the effect of panaxytriol on the cytotoxicity of mitomycin C (MMC) against a human gastric carcinoma cell line, MK-1, was investigated. The combination of a subthreshold concentration of MMC and panaxytriol produced a significant cytotoxic effect, which indicates that the effects of panaxytriol and MMC are synergistic. A synergistic effect was observed when MK-1 cells were treated with the mixture of MMC and panaxytriol or treated with MMC followed by panaxytriol. In contrast, when MK-1 cells were exposed to panaxytriol and then to MMC, only an additive effect was induced. With the aim of finding a possible mechanism, the effect of panaxytriol on the accumulation of MMC into the MK-1 cells was examined. Cellular concentrations of MMC were measured by high-performance liquid chromatography (HPLC). When MK-1 cells were treated with a mixture of panaxytriol and MMC or first with MMC and then with panaxytriol, the cellular level of MMC was significantly higher than that in MK-1 cells treated with MMC alone, but no significantly increased accumulation was found when MK-1 cells were treated with panaxytriol followed by MMC. These results suggest that synergistic effects of panaxytriol and MMC may be induced by acceleration of the effect of MMC on cellular accumulation by panaxytriol. In addition, they suggest that the enhanced accumulation of MMC in MK-1 cells treated with panaxytriol can probably be attributed to the decreased fluidity of the cell membrane caused by panaxytriol.

In vitro effect of lonidamine on the cytotoxicity of mitomycin C and BCNU in human colon adenocarcinoma cells

The ability of lonidamine (LND), an energolytic derivative ofindazole carboxylic acid, to modulate the cytotoxic activity of mitomycin C (MMC) and l,3,bis(2-chloroethyl)- l-nitrosourea (BCNU) was investigated in two human adenocarcinoma cell lines (LoVo and HT29) expressing different sensitivity profiles to the drugs. After a 1-h treatment with MMC or BCNU, cells were postincubated for 24 h with 150–225 μM LND. In the LoVo cells, a synergistic interaction between LND and MMC or BCNU was observed at both LND concentrations. In HT29 cells, only additive effects of the drugs given in sequence were seen. Flow cytometric analysis indicated that LND was generally able to stabilise the cell cycle perturbations induced by MMC and BCNU in the two cell lines. The ability of LND to potentiate anticancer drug activity, and the consideration that LND causes side-effects different from those of conventional antitumour drugs, make this compound an attractive candidate for multidrug combination therapy in colon cancer.

Potentiation of cytotoxicity of mitomycin C by a polyacetylenic alcohol, panaxytriol

Potentiation of cytotoxicity of mitomycin C by a polyacetylenic alcohol, panaxytriol

Effect of artificial electron acceptors on the cytotoxicity of mitomycin C and doxorubicin in human lung tumor cells : Keizer HG, De Leeuw SJ, Van Rijn J, Pinedo HM, Joenje H. Institute of Human Genetics, Free University, 1007 MC Amsterdam. Eur J Cancer Clin Oncol 1989;25:1113-8

Effect of artificial electron acceptors on the cytotoxicity of mitomycin C and doxorubicin in human lung tumor cells : Keizer HG, De Leeuw SJ, Van Rijn J, Pinedo HM, Joenje H. Institute of Human Genetics, Free University, 1007 MC Amsterdam. Eur J Cancer Clin Oncol 1989;25:1113-8

Addition of a hypoxic cell selective cytotoxic agent (mitomycin C or porfiromycin) to Fluosol-DA ®/carbogen/radiation

In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O 2/5% CO 2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and hypoxic tumor subpopulations can markedly increase the tumor cell kill achieved by radiation.

Effect of artificial electron acceptors on the cytotoxicity of mitomycin C and doxorubicin in human lung tumor cells

The cytotoxicities of mitomycin C (MMC) and doxorubicin (DOX) have been proposed to depend on intracellular reduction by reduced flavoproteins. We investigated whether MMC- and DOX-induced cytotoxicity could be inhibited by competing for electrons from reduced flavoproteins by the artificial electron acceptors phenazine methosulfate (PMS), menadione (MEN) and methylene blue (MB). In intact SW- 1573 human lung tumor cells these compounds proved to be excellent electron acceptors, as judged from their capacity to induce high levels of cyanide-resistant respiration. In addition, PMS, MEN and MB were found to decrease the cytotoxicity of MMC, by 90, 63 and 29%, respectively, at concentrations that were themselves completely nontoxic. In contrast, DOX cytotoxicity was not detectably affected. These results suggest that in SW- 1573 cells flavoprotein-mediated bioreduction is required for the cytotoxic effect of MMC, but not for that of DOX.

Glutathione levels and cytotoxicity of a thiol activated alkylating agent in human and mouse cells

The effects of cellular GSH levels on the cytotoxicity of MNNG and mitomycin C were examined in normal and BSO treated mouse C3H10T 1 2 cells. MNNG was less cytotoxic in the GSH depleted cells (<10% of normal) whereas the cytotoxicity of mitomycin C was not influenced by thiol status. This is compatible with the alkylating agent MNNG requiring thiols for activation to the methylating electrophile. Conversely, thiols have little if any effect in modulating the activity of mitomycin C. When naturally thiol deficient human fibroblasts were compared with BSO treated fibroblasts depleted to a similar GSH level (<10% of normal), only the BSO depleted cells were less effected by MNNG. The GSH deficient cells showed the same MNNG dose response as normal human fibroblasts. These studies indicate that a naturally acquired thiol status and an equivalent induced thiol status need not behave the same and this needs consideration when evaluating the role of thiols in influencing cellular response to chemotherapeutic agents.