Search

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.

Some factors affecting the cytotoxicity of porfiromycin (PM), an analogue of mitomycin C (MMC), were investigated in suspension cultures of wild-type (AA8-4) and repair-deficient (UV-20) Chinese hamster ovary cells. Oxygen was an important modulator of PM toxicity in AA8-4 cells. The aerobic toxicity was significantly less, and toxicity under extremely hypoxic conditions was significantly greater for PM than MMC. Porfiromycin cytotoxicity at intermediate O2 levels was similar to that observed previously for MMC. While the aerobic/hypoxic ratio was greater for PM than MMC, survival at intermediate oxygen concentrations could limit the therapeutic utility of these drugs as adjuncts to radiotherapy. Ascorbic acid was found to increase the aerobic, but not hypoxic, cytotoxicity of PM in AA8-4 cells, as was observed previously for MMC. Investigation of various exposure times and drug concentrations revealed that drug toxicity for both aerobic and hypoxic cells was dependent on the product of drug concentration and time, and that the aerobic/hypoxic differential observed in AA8-4 cells was constant over a broad range of exposure conditions. The sensitivity of UV-20 cells was also a linear function of concentration and time, but no aerobic/hypoxic differential was observed in these cells. It is suggested that the sensitivity of UV-20 to PM and MMC, and its lack of an hypoxic/aerobic differential could result from lethality being due to a different lesion than in wild-type cells.

We have demonstrated previously that dicoumarol (DIC) increased the generation of reactive metabolites from mitomycin C (MC) in EMT6 cells under hypoxic conditions in vitro. This increased reaction rate was associated with an increased toxicity of MC to hypoxic EMT6 cells. In contrast, aerobic cells treated with DIC in vitro were protected from MC toxicity. We now demonstrate that DIC sensitizes EMT6 cells to two MC analogues, porfiromycin (POR) and the 7-N-dimethylaminomethylene analogue of mitomycin C (BMY-25282), in hypoxia and protects cells from these agents in air, despite the fact that POR is preferentially toxic to hypoxic cells and BMY-25282 is preferentially toxic to aerobic cells. In contrast, DIC increases menadione cytotoxicity in both air and hypoxia and has no effect on the cytotoxicity of Adriamycin. We have also shown previously that the preferential toxicity of POR to hypoxic cells is associated with an increased rate of drug uptake. In the present study, DIC had no measurable effect on the uptake of [3H]POR but increased the extent of efflux of this agent. MC-induced DNA cross-links, which have been proposed as the lesions responsible for the lethality of MC, are decreased by DIC in air and increased by DIC in hypoxia, in concert with the observed modifications of MC cytotoxicity by DIC. However, in aerobic cells treated with DIC and MC, the decrease in DNA interstrand cross-links is not directly associated with a decrease in cytotoxicity. L1210 cells, which have no measurable quinone reductase activity, demonstrate increased toxicity when treated with DIC and MC in hypoxia, as observed with EMT6 cells. Unlike EMT6 cells, however, L1210 cells are not protected by DIC from MC toxicity in air. Taken together, these findings suggest that DIC is altering the intracellular metabolism of MC and that quinone reductase or another, unidentified, enzyme sensitive to DIC may be involved in activating MC to a toxic product in aerobic EMT6 cells.

Hypoxic cells may limit the curability of solid tumors by conventional chemotherapeutic agents and radiotherapy. Agents which are preferentially toxic to cells with low oxygen contents could therefore be useful as adjuncts to the regimens now used to treat these cancers. To date, the best agent of this type that we have tested is porfiromycin. Porfiromycin is similar to mitomycin C in its toxicity to hypoxic EMT6 cells in vitro but has much less toxicity than mitomycin C to well-oxygenated EMT6 cells. EMT6 cell sonicates reduce mitomycin C and porfiromycin to reactive electrophiles at similar rates under hypoxic conditions, a finding that correlates with cytotoxicity, whereas the rate of production of reactive species from both drugs is very slow under aerobic conditions. We also show that porfiromycin is capable of killing hypoxic radiation-resistant cells in solid EMT6 tumors. Appropriate regimens combining porfiromycin (which preferentially kills hypoxic cells) and radiation (which preferentially kills aerated cells) may therefore be especially efficacious for the treatment of solid tumors.

The mechanism of uptake and efflux of porfiromycin (PFM) by HCT 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to [14C]PFM showed one fast and one slow phase of linear increase. The initial phase of PFM uptake was not saturable with external drug concentrations from 2 to 100 microM. PFM accumulation was temperature dependent with a temperature coefficient (Q10 24-37 degrees C) of 2.3 +/- 0.3. PFM uptake was not affected either by individual inhibitors such as 1 mM 2,4-dinitrophenol, sodium azide, iodoacetic acid, ouabain, 0.02 mM oligomycin, p-hydroxylmercuribenzoate, 0.2 mM N-ethylmaleimide, or by combinations of inhibitors. PFM uptake did not demonstrate competitive inhibition by unlabeled PFM and mitomycin C. Efflux of cellular radioactivity was not affected by the above mentioned inhibitors or by verapamil, diltiazem, or trifluoperazine. Only aliphatic alcohols accelerated the initial influx rate. The RBC, however, only exhibited the initial fast accumulation of [14C]PFM, and all the 14C accumulated by RBC was exchangeable. These data demonstrate that the uptake and the efflux of PFM in HCT 116 cells and RBC comprise a passive diffusion process.

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)