Interstrand DNA-DNA and DNA-protein cross-links by a new antitumor antibiotic, FK973, in L1210 cells

In our previous study FK973, a novel, substituted dihydrobenzoxazine (11-acetyl-8-carbamoyloxymethyl-4-formyl-14-oxa-1,11-diazatetracyclo+ ++ [7.4.1.0(2,7).0(10,12)]tetradeca-2,4,6-trien-6,9-diyl diacetate), had potent cytotoxic and antitumor effects on murine tumors and human tumors in in vitro and in vivo experiments. In the present study the mechanism(s) of the in vitro cytotoxic effects of the drug on tumor cells were studied. After 1-h exposure of L1210 murine leukemia cells to the drug, the concentration of FK973 required to inhibit cell growth by 50% was approximately 1 microM, which was threefold more potent than the concentration of mitomycin C required. DNA synthesis was selectively inhibited in the cells treated with FK973. Alkaline elution analyses showed that FK973 formed concentration- and time-dependent interstrand DNA-DNA and DNA-protein cross-links in the cells. On the other hand, no DNA single-strand breaks were observed in the cells treated with FK973. When isolated nuclei of L1210 were exposed to FK973 for 1 h, FK973 did not form detectable interstrand DNA-DNA cross-links. We propose that FK973 is activated in the cytoplasm of cells, and forms interstrand DNA-DNA and DNA-protein cross-links which may be important for the induction of its cytotoxicity.     

In vivo DNA cross-linking by cyclophosphamide: comparison of human chronic lymphatic leukemia cells with mouse L1210 leukemia and normal bone marrow cells

Alkaline elution was done on a variety of cells following cyclophosphamide (CY) treatment in vivo. Cells used were L1210 leukemia, normal mouse bone marrow, and peripheral blood cells obtained from a patient with chronic lymphatic leukemia (CLL). Endpoints used were determination of single strand breaks, DNA-DNA interstrand and DNA-protein cross-links. After treatment of mice with CY (4 mg/mouse), low levels of single strand breaks were observed in both L1210 leukemia and CDF1 normal bone marrow. When a patient with CLL was treated with CY (750 mg/m2), no evidence of single strand breaks could be demonstrated. Maximum levels of DNA-DNA interstrand cross-links were observed in mice 2 h after injection of CY for the L1210 leukemia cells [175 +/- 25 rad-equivalents (req)] and at 4 h for the CDF1 normal bone marrow cells (47 +/- 9 req). In human CLL, maximum levels were observed 12 h after injection of CY. Peak levels of DNA-DNA interstrand cross-links were approximately 4-fold lower in CDF1 normal bone marrow cells than in L1210 leukemia. The frequencies measured in human CLL cells were relatively low at any timepoint (mean at 12 h = 36 req). Maximum levels of DNA-protein cross-links were observed 4 h after injection of CY (4 mg/mouse) for both L1210 leukemia [123 req (mean)] and normal bone marrow cells [50 req (mean)]. DNA-protein cross-links were measurable in CLL at timepoints later than 4 h after the start of injection of CY. In order to obtain equitoxicity between L1210 leukemia and CDF1 normal bone marrow cells, about 18-fold higher doses of CY had to be given than in the case of the normal bone marrow cells. In contrast, only 4-fold higher doses had to be given to the normal bone marrow to obtain equivalent peak levels of DNA-DNA interstrand cross-links.     

Involvement of DT-diaphorase (EC 1.6.99.2) in the DNA cross-linking and sequence selectivity of the bioreductive anti-tumour agent EO9.

The chemistry of the mitomycin C-related drug indoloquinone EO9 would suggest that its mechanism of action is likely to involve DNA damage after reductive activation. The ability of this agent to induce DNA damage in intact cells has been examined using alkaline filter elution. After treatment with pharmacologically relevant concentrations of EO9, both DNA strand breaks and interstrand cross-links were detected in rat Walker tumour cells and human HT29 colon carcinoma cells. These cell lines express relatively high levels of DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase), which is believed to be involved in EO9 activation. The extent of DNA damage was increased by approximately 30-fold under hypoxia in BE colon carcinoma cells that express non-functional DT-diaphorase, but this dramatic hypoxia enhancement was not seen in HT-29 cells. These data are consistent with cytotoxicity studies that indicate that DT-diaphorase appears to be important in EO9 activation under aerobic conditions, but other enzymes may be more relevant under hypoxia. The involvement of DT-diaphorase in DNA damage induction was further investigated using cell-free assays. DNA cross-links were detectable in plasmid DNA co-incubated with EO9, cofactor and DT-diaphorase but not in the absence of this enzyme. In contrast, using a Taq polymerase stop assay, monofunctional alkylation was detected in plasmid DNA without metabolic activation, although the sequence selectivity was altered after reduction catalysed by DT-diaphorase.     

Chemosensitivity correlation between the primary tumors and simultaneous metastatic lymph nodes of patients evaluated by DNA synthesis inhibition assay

The chemosensitivities of primary tumors (PT) and simultaneous metastatic lymph nodes (MN) to mitomycin C (MMC), 5-fluorouracil (5-FU), Adriamycin (ADR) (doxorubicin; Adria Laboratories, Columbus, OH), carboquone (CQ), or cisplatin (CDDP) were assessed in a group of 29 patients (11 gastric, 8 colorectal, 4 breast, and 6 other cancers) by a DNA synthesis (3H-thymidine incorporation) inhibition assay. PT and MN from the same patient showed heterogeneity in chemosensitivity. MN were more sensitive to the agents than PT. PT were sensitive to 5-FU, whereas MN were sensitive to CDDP. An analysis of the sensitivity correlations showed that the sensitivities of PT to MMC, 5-FU, CQ, and CDDP correlated with each other, but ADR sensitivity correlated with only CQ sensitivity. The sensitivities of MN correlated with each other, except for those to ADR and CDDP. In contrast, MMC, ADR, or CQ sensitivity showed a correlation between PT and MN. These results suggest that patients should be treated according to the sensitivity of the target lesion. However, if the sensitivity assay is not available, the sensitivity correlation may be useful when choosing the agent. It also may be important that ADR sensitivity does not correlate with the sensitivities of other agents.     

Interactions of mitomycin C with mammalian DNA detected by alkaline elution

The antitumor antibiotic mitomycin C (MMC) was studied in vitro using L1210 leukemia and 8226 human myeloma cells. Cytotoxicity was evaluated by colony formation in soft agar, and DNA damage was analyzed using alkaline elution filter assays. The purposes of these studies were: (a) to characterize the time course of MMC-DNA damage; (b) to characterize the type of DNA damage [DNA-DNA interstrand cross-links (ISC), DNA-protein cross-links (DPC), single and double strand breaks (SSBs, DSBs)]; and (c) to correlate this damage with cytotoxicity in vitro. Colony-forming assays showed the D0 value for 1 h MMC to be 15.0 microM for L1210 cells and 17 microM for 8226 cells. Alkaline elution studies showed that dose-dependent ISCs and DPCs formed rapidly following MMC exposure. Removal of cross-links was delayed, with only 50% repaired 32 h after exposure. There was a good correlation between ISCs and cytotoxicity in dose-response studies in each cell line. ISCs appeared to comprise most of the MMC-DNA lesions in both cell lines. No DNA SSBs or DSBs were observed following MMC exposure. Nuclei isolated from both cell lines and exposed to MMC produced less MMC alkylation than whole cells but, again, no strand breaks were evident. These results demonstrate that MMC is principally an alkylating agent when used at pharmacological (cytotoxic) concentrations in vitro. The lack of evidence for DNA strand breaks discounts a significant role for putative quinone-generated oxygen free radicals in the production of MMC cytotoxicity.     

Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.     

Influence of WR2721 on DNA cross-linking by nitrogen mustard in normal mouse bone marrow and leukemia cells in vivo

In previous studies we showed that WR2721 enhanced nitrogen mustard (HN2) cytotoxicity to AKR mouse leukemia. Simultaneously, it protected normal bone marrow from drug cytotoxicity. In this study, the method of alkaline elution was used to measure the modifications of HN2 induced DNA damage in the same animal model. In leukemia cells, no significant differences in DNA-DNA interstrand cross-links were observed between mice treated with WR2721 and HN2 and mice treated with HN2 alone. In normal bone marrow, DNA-DNA cross-linking was decreased by about 50% in mice treated by WR2721 and HN2. The difference was significant (P = 0.01). In leukemia, half-time of repair was 73 min for HN2 and 83 for WR2721 and HN2 treated mice, while in normal bone marrow, respectively, 73 and 64 min were calculated. WR2721 modulation significantly decreases DNA-protein cross-links in leukemia cells (P less than 0.05). This decrease was observed for early as well as for late DNA-protein cross-links. In normal bone marrow cells, only early DNA-protein cross-links were decreased. The meaning of this observation is unclear.     

Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline

Hepatic DNA damage induced by the pyrrolizidine alkaloid monocrotaline was evaluated following i.p. administration to adult male Sprague-Dawley rats. Animals were treated with various doses ranging upward from 5 mg/kg, and hepatic nuclei were isolated 4 hr later. Hepatic nuclei were used as the DNA source in all experiments. DNA damage was characterized by the alkaline elution technique. A mixture of DNA-DNA interstrand cross-links and DNA-protein cross-links was induced. Following an injection of monocrotaline, 30 mg/kg i.p., DNA-DNA interstrand cross-linking reached a maximum within 12 hr or less and thereafter decreased over a protracted period of time. By 96 hr postadministration, the calculated cross-linking factor was no longer statistically different from zero. No evidence for the induction of DNA single-strand breaks was observed, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. These DNA cross-links may be related to the hepatocarcinogenic, hepatotoxic, and/or antimitotic effects of monocrotaline.     

Effects of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin and structural analogues on DNA in HT-29 human colon carcinoma cells

The potent Adriamycin (ADR) analogue, 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA), produces DNA-DNA cross-links in human and murine tumor cells. The cellular pharmacology of CMA, its derivative, 5-imino-3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (ICMA), 3'-(4-morpholinyl)-3'-deaminoadriamycin (MA), and ADR was evaluated in HT-29 human colon carcinoma cells to determine the structural requirements for the cross-linking activity of CMA, and the role of this activity in the antitumor effect of this agent. CMA was 50-fold more cytocidal than ICMA to HT-29 cells, 300-fold more toxic than MA, and 150-fold more potent than ADR. Both CMA and ICMA produced DNA-DNA cross-links in HT-29 cells but, consistent with its reduced cytotoxicity, the imino derivative was 30-fold less active than CMA. No DNA-DNA cross-links were observed with MA or ADR. CMA also showed cross-linking activity in isolated HT-29 nuclei, indicating that cytoplasmic activation was not required for this effect. Both CMA and ICMA produced cross-links in isolated lambda-phage DNA with CMA being 40-fold more active than the imino derivative, and this activity was unchanged in the presence or absence of a reducing agent. While MA and ADR produced DNA strand breaks in HT-29 cells, this damage was not observed with CMA and ICMA. This study indicates that the potent antitumor activity of CMA may be related to its ability to induce DNA cross-links, which can occur without the need for metabolic activation. The cyanide group appears to be essential for cross-linking and the quinone group may also be involved, but by a mechanism unrelated to its reduction.     

Different Effects of FK317 on Multidrug-resistant Tumor in vivo and in vitro

FK317, a novel substituted dihydrobenzoxazine, was examined for antitumor effects on multidrug-resistant (MDR) tumor cells in vitro and in vivo . In nude mice, FK317 markedly inhibited the growth of s.c. implanted KB-V1 vinblastine (VLB)-resistant human epidermal carcinoma KB cells, as well as the parent cells (KB-3-1). However, KB-V1 showed much greater resistance to FK317 than to VLB and adriamycin (ADM) in the in vitro study. This resistance was reversed by the addition of verapamil, whereby intracellular accumulation of FK317 in the KB-V1 cells was also decreased. After incubation of FK317 in human and mouse blood, it was shown to be rapidly metabolized to a monodeacetylated form, and slowly metabolized further to a dideacetylated form. With the removal of the acetyl groups from FK317, resistance indexes in KB-V1 and SBC-3/ADM, ADM-resistant human lung carcinoma, decreased. In addition, photolabeling of P-glycoprotein with [³H]azidopine in KB-V1 plasma membrane was completely inhibited by FK317, but not by the deacetylated metabolites. These results indicate that FK317 is metabolized to deacetylated forms, which do not bind to P-glycoprotein and are incorporated into MDR cells, causing cytotoxic effects.     

In vivo inhibitory effect of anticancer agents on human pancreatic cancer xenografts transplanted in nude mice

Pancreatic cancer is one of the neoplasms resistant to chemotherapy. In the present study human pancreatic cancer xenografts (3 adenocarcinomas and 1 cystoadenocarcinoma) were subcutaneously transplanted in nude mice and after the tumors grew to 100-300 mm3, the mice were intraperitoneally administered with mitomycin C (MMC), adriamycin (ADR), 5-fluorouracil (5-FU), carboquone (CQ), cisplatinum (CDDP), nimustine chloride (ACNU) or DWA2114R at 1/3 LD50 on days 0.4, and 8. The tumor sizes on day 12 were compared with those on day 0. MMC and CQ significantly inhibited the tumor growth of 3 lines, and ACNU, CDDP and ADR inhibited the growth of 1 line. Further, 5-FU, futrafur, carmofur, UFT and L-phenylalanine mustard (L-PAM) were orally administered to mice into which 1 adenocarcinoma line had been transplanted. While none of fluoropyrimidines inhibited tumor growth, L-PAM at 4 mg/kg significantly inhibited growth, although it was accompanied by severe body weight loss. In the present study several agents significantly inhibited tumor growth, but none of them could induce the regression of the tumor when used singly. These results suggest that CQ, ACNU, CDDP and L-PAM may be applied to the chemotherapy of pancreatic cancer. However, the effect of a single agent is restricted and the development of new combination treatments is urgently required.     

Mechanisms of toxicity of hepsulfam in human tumor cell lines

1,7-Heptanediol disulfamate (hepsulfam, NSC 329680) is a new anti-cancer agent which is currently undergoing phase I clinical trials. The mechanism of action of this compound is not clear at this time. We have recently shown that hepsulfam was more toxic to L1210 leukemia cells than was busulfan. Consistent with the difference in toxicity, we found that hepsulfam induced DNA interstrand cross-links in L1210 mouse leukemia cells, whereas busulfan did not. In the present study, we have found that hepsulfam was more cytotoxic to two human leukemia cell lines (HL-60 and K562) and to two human colon carcinoma cell lines (BE and HT-29) than was busulfan. As in L1210 cells, hepsulfam induced a higher level of DNA interstrand cross-links than busulfan. Both compounds induced DNA-protein cross-links. Hepsulfam was also more cytotoxic to the human leukemia cell lines when the concentrations were reduced 10-fold and the duration of drug exposure was increased to 12-h This more accurately reflects the drug exposures that human leukemia cells may encounter in vivo. Under these 12-h drug exposures, hepsulfam was still able to form DNA interstrand and DNA-protein cross-links, whereas busulfan was only able to form DNA-protein cross-links. These results show that busulfan and hepsulfam react with DNA differently and that hepsulfam is a more potent cytotoxic agent.     

Indoloquinone EO9: DNA interstrand cross-linking upon reduction by DT-diaphorase or xanthine oxidase.

We report DNA interstrand cross-linking caused by the anti-tumour indoloquinone EO9 following reductive activation with purified rat liver DT-diaphorase or xanthine oxidase. Reduction was a necessary event for cross-linking to occur. DNA cross-link formation by EO9 following DT-diaphorase reduction was completely inhibited by addition 10 microM dicoumarol, whereas only a minor effect of dicoumarol on xanthine oxidase-mediated DNA cross-linking by EO9 was observed. DNA cross-linking was pH dependent, with increasing cross-link formation from pH 5.5 to 7.0 for both DT-diaphorase and xanthine oxidase mediated reactions. Also, conversion of EO9 upon reduction was pH dependent. However, in contrast to DNA cross-linking, conversion rates of EO9 decreased at higher pH. EO9 was shown to be more efficient in DNA cross-linking than mitomycin C under identical conditions, using both DT-diaphorase and xanthine oxidase reductive activation at pH 5.5 and 7.0. This study indicates that the anti-tumour activity of EO9 may be at least partly mediated by interstrand DNA cross-link formation, and that various reducing enzymes may be important for activation of EO9 in vitro and in vivo.     

Inhibition of cis-diamminedichloroplatinum (II)-induced DNA interstrand cross-link removal by 7-ethyl-10-hydroxy-camptothecin in HST-1 human squamous-carcinoma cells

The combination of cis-diamminedichloroplatinum(II) (CDDP) and 7-ethyl-10-[4-(I -piperidino)-I -piperidino]carbonyloxycamptothecin (CPT-I I), a topoisomerase-l inhibitor, has been shown to be synergistic in vitro and clinically active against several human cancers refractory to chemotherapy. To elucidate the mechanism of the synergistic cytotoxicity of CDDP and 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of CPT-11, we studied the interaction of these agents using an HST-I human squamous-carcinoma cell line. Cells were exposed to the IC₅₀ concentration of SN-38 (5.0 ng/ml) for I hr and various concentrations of CDDP for 1 hr in several different treatment schedules. SN-38 augmented the anti-tumor activity of CDDP in all schedules, with maximal synergy observed with simultaneous administration. Evaluation of the kinetics of the removal of DNA interstrand cross-links, measured by alkaline elution, showed significant reduction of this removal in the cells exposed to SN-38 and CDDP, as compared with the cells exposed to CDDP alone. No differences, however, were found in the initially attained level of DNA interstrand cross-links induced by CDDP between these cells. Moreover, the intracellular accumulation of platinum measured by atomic-absorption spectrophotometry, was virtually identical between these cells. These results indicate that SN-38 can modulate the removal of platinum-DNA adducts, thereby potentiating the cytotoxicity of CDDP, suggesting a critical role for topoisomerase I in the repair of DNA interstrand cross-links. © 1995 Wiley-Liss Inc.     

Establishment by Adriamycin Exposure of Multidrug-resistant Rat Ascites Hepatoma AH130 Cells Showing Low DT-diaphorase Activity and High Cross Resistance to Mitomycins

A resistant subline (AH130/5A) selected from rat hepatoma AH130 cells after exposure to adriamycin (ADM) showed remarkable resistance to multiple antitumor drugs, including mitomycin C (MMC) and porflromycin (PFM). PFM, vinblastine (VLB), and ADM accumulated in AH130/5A far less than in the parent AH130 (AH130/P) cells. AH130/5A cells showed overexpression of P-glycoprotein (PGP), an increase in glutathione S-transferase activity, and a decrease in DT-diaphorase and glutathione peroxidase activity. The resistance to MMC and VLB of AH130/5A cells was partly reversed by H-87, an inhibitor of PGP. Buthionine sulfoximine, an inhibitor of glutathione synthase, did not affect the action of MMC. tert -Butylhydroquinone induced DT-diaphorase activity, increased PFM uptake, and enhanced the growth-inhibitory action of MMC in AH130/5A cells. Dicumarol, an inhibitor of DT-diaphorase, decreased PFM uptake and reduced the growth-inhibitory action of MMC in AH130/P cells. These results indicated that the adriamycin treatment of hepatoma cells caused multifactorial multidrug resistance involving a decrease in DT-diaphorase activity.     

Comparison of DNA damage by methylmelamines and formaldehyde

The cytoxicity and DNA damaging activity of S9-activated hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were compared with suspected active metabolites in mouse leukemia L1210 cells. The presence of semicarbazide hydrochloride did not alter the cytotoxicity of S9-activated HMM and PMM or that of their hydroxylated analogs monomethylolpentamethylmelamine (MPM) and trimethyloltrimethylmelamine (TTM), which have been suggested as active metabolites. Following treatment of L1210 cells with high concentrations of activated HMM and PMM, there were no DNA single-strand breaks or interstrand cross-links observed by DNA alkaline elution and only a low frequency of DNA-protein cross-links. Formaldehyde (FA) at nonlethal concentrations caused far greater DNA-protein cross-linking. In contrast, the polyfunctional TTM produced both DNA-protein cross-linking and DNA interstrand cross-linking. The cytotoxicities of HMM and PMM were found unlikely to be related to extracellular or intracellular release of FA.     

In vivo and in vitro evaluation of the alkylating agent carmethizole

Carmethizole, a novel bis-carbamate alkylating agent, was evaluated in vitro for potential mechanisms of interaction with DNA and in vivo for spectrum and degree of antitumor activity. In vitro, the concentration of carmethizole required to produce a 50% reduction in clonogenic cell survival was identical in O6-alkylguanine DNA alkyltransferase-positive and -negative human cell lines. The CHO cell line UV4, hypersensitive to mono- and bifunctional alkylating agents, was 37-fold more sensitive to carmethizole than normal cells. The UV5 cell line, which is not hypersensitive to cross-linkers, was 13-fold more sensitive to carmethizole than normal cells. Alkaline elution studies in L1210 cells exposed to carmethizole showed the presence of DNA-protein and DNA-DNA cross-links but not DNA strand breaks. These data suggested that the interaction of carmethizole with DNA produces monoadducts, DNA-protein, and DNA-DNA interstrand cross-links at several sites. In vivo, carmethizole was not cross-resistant with 1,3-bis(2-chloroethyl)-1-nitrosourea or Cytoxan as determined by testing against P388 leukemias resistant to the latter 2 agents. Carmethizole activity was similar to that of melphalan across the murine solid tumor panel, which consisted of B16 melanoma; colon adenocarcinomas 11a, 26, and 36; and the KHT sarcoma. Carmethizole, Cytoxan, and melphalan were all active and had comparable activity against the HCT-8 and MX-1 human tumor xenografts. The in vivo spectrum of activity and efficacy of carmethizole was similar to that of melphalan.     

Schedule-dependent reversion of acquired cisplatin resistance by 5-fluorouracil in a newly established cisplatin-resistant HST-1 human squamous carcinoma cell line

In vitro continuous stepwise exposure of HST-I human squamous carcinoma cell line to cisplatin (CDDP) for 12 months resulted in a 3.5-fold stably resistant subline designated HST-I/CPO.2. Compared with parental cells, this cell line showed a 1.8-fold increase in cellular glutathione (GSH) and a 50% reduction in initial numbers of DNA interstrand cross-links (ICLs), despite similar levels of intracellular platinum accumulation. Evaluation of the kinetics of DNA ICL removal at nearly equivalent levels of DNA ICL formation indicated that HST-I/CPO.2 cells appeared to remove DNA ICLs more rapidly than do HST-I parental cells. Thus, both elevated cellular GSH and increased DNA repair capacity would be the major factors contributing to CDDP resistance. Pretreatment of HST-I/CPO.2 cells with 5-FU, with drug-free intervals of 24 to 48 hr before exposure to CDDP, completely reversed CDDP resistance, or even increased the sensitivity to a level greater than that of parental cells, whereas the opposite sequence had no effect on resistance. In parallel with augmentation of the cytotoxicity, the levels of cellular GSH were significantly reduced over 48 hr by 5-FU pretreatment. However, depletion of cellular GSH using buthionine sulfoximine resulted in partial reversal of CDDP resistance, indicating that reduction of cellular GSH alone is not sufficient for complete reversal of CDDP resistance. Our data, together with evidence that 5-FU modulates the repair of platinum-DNA cross-links, suggest that schedule-dependent, complete reversal of CDDP resistance by 5-FU might be attributed to its inhibitory effects on both GSH levels and the repair of platinum-DNA adducts. Thus, optimization for the drug administration schedule is important when aiming at therapeutic synergy and circumvention of acquired CDDP resistance. © 1996 Wiley-Liss, Inc.     

Characterization of a human bladder cancer cell line selected for resistance to mitomycin C

This study describes characteristics of a human bladder cancer cell line J82/MMC that is 6-fold more resistant to mitomycin C (MMC) than the parental cells. The J82/MMC sub line was isolated by repeated continuous exposures of the J82/WT cells to increasing concentrations of MMC. The J82/MMC cell line showed (1) collateral sensitivity to taxol, 5-FU and topoisomerase II inhibitors; and (2) cross-resistance to cisplatin, melphalan and MMC analogues BMY 25282 and BMY 25067. Levels of two key MMC activation enzymes, NADPH cytochrome P450 reductase and DT-diaphorase, were significantly lower in J82/MMC cells compared with J82/WT, suggesting that lower sensitivity of J82/MMC cells to MMC may result from deficient drug activation. Further support is indicated by: 1) reduction in the differential in toxicity between the 2 cell lines by BMY 25282; and 2) a higher effect of DT-diaphorase inhibitor dicumarol on the wild-type cells compared with J82/MMC. Although glutathione (GSH) levels did not differ in these cells, a small but significant increase in GSH transferase (GST) activity was noticed in J82/MMC cells. GST inhibitor ethacrynic acid significantly enhanced MMC cytotoxicity in the J82/MMC cell line. A small but significant increase in the level of anti-oxidative enzyme catalase, but not GSH peroxidase, was also observed in J82/MMC cell line compared with J82/WT. Thus, the possibility that relatively lower sensitivity of J82/MMC cells to MMC may result from reduced oxygen radical generation cannot be ruled out. MMC-induced DNA interstrand cross-linking was markedly lower in the J82/MMC cell line compared with J82/WT. Our results suggest that the MMC resistance in the J82/MMC cell line may be multifactorial.     

Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells.

The role of DT-diaphorase in bioreductive activation of mitomycin C was examined using HT-29 and BE human carcinoma cells which have high and low levels of DT-diaphorase activity, respectively. HT-29 cells were more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrand cross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links could be inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolism of mitomycin C by HT-29 cell cytosol was pH dependent and increased as the pH was lowered to 5.8, the lowest pH tested. Metabolism of mitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosol or by the inclusion of dicoumarol. Little metabolism was detected in BE cytosols. When purified rat hepatic DT-diaphorase was used, metabolism of mitomycin C increased as the pH was decreased and could be detected at pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C was NADH dependent and inhibited by dicoumarol or by prior boiling of enzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could be detected. Metabolism of mitomycin C by purified HT-29 DT-diaphorase was also dicoumarol inhibitable and pH dependent. The major metabolite formed during metabolism of mitomycin C by HT-29 cytosol, purified HT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diaminomitosene. These data suggest that two-electron reduction of mitomycin C by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.