Lidocaine potentiation of bleomycin A2 cytotoxicity and DNA strand breakage in L1210 and human A-253 cells

The survival of cultured L1210 cells exposed to bleomycin A2 (BLM A2) was markedly decreased by coincubation with the local anesthetic lidocaine. The potentiation occurred with concentrations of lidocaine that were nontoxic and was dependent upon both the concentration of lidocaine and BLM A2. A 1000-fold decrease in survival was seen with a 1-h exposure to 8 mM lidocaine and 10 microM BLM A2 compared to incubation with 10 microM BLM A2 alone. Prior exposure to lidocaine did not markedly alter BLM A2 cytotoxicity, while treatment with lidocaine immediately after BLM A2 exposure did, suggesting that increased cellular content of BLM A2 was not the mechanism of enhancement. Furthermore, lidocaine reduced the total amount of cell-associated radioactivity seen after incubation with [3H]BLM A2. The enhancement in L1210 cell cytotoxicity with lidocaine was not specific for the C- and N-terminal moieties of the BLM molecule. Other DNA-interacting antitumor agents, such as etoposide and mitomycin C, did not exhibit biologically significant alterations in their cytotoxicity when coincubated with lidocaine, although cis-diamminedichloroplatinum was significantly more toxic in the presence of lidocaine. The potentiation of BLM A2 cytotoxicity was not unique to murine tumor cells, since it was also seen with cultured human head and neck carcinoma (A-253) cells. Lidocaine did not increase directly BLM A2-induced breakage of DNA in vitro as measured by loss of form I pAT 153 DNA, but it did increase BLM A2-induced DNA strand breaks in intact L1210 cells coincubated with lidocaine and BLM A2. Exposure of L1210 cells to lidocaine after BLM A2 treatment also greatly increased DNA breakage consistent with possible inhibition of DNA repair. In addition, a modest reduction in the in vitro inactivation of BLM A2 by BLM hydrolase was found with lidocaine. We propose that inhibition of BLM metabolism and repair of BLM-induced DNA damage by lidocaine may have a role in the enhanced cytotoxicity.     

Pre-treatment of a human T-lymphoblastoid cell line with L-asparaginase reduces etoposide-induced DNA strand breakage and cytotoxicity.

The effect of L-asparaginase (L-asp) pre-treatment on etoposide-induced DNA strand breakage and cytotoxicity was investigated. In a T-lymphoblastoid cell line, Molt 4, etoposide-induced DNA strand breaks, DNA-protein cross-links and cytotoxicity were reduced by pre-treatment with L-asp for 15 hr, but it did not cause these changes in a promyelocytic-leukemia cell line, HL-60, which is less sensitive than Molt 4 to L-asp. However, pre-treatment of Molt 4 cells with L-asp did not significantly alter the accumulation of [3H]-etoposide. Cell-cycle analyses showed an increase in G1-phase cells, a significant decrease in both S-phase cells and G2/M-phase cells pre-treated with L-asp in Molt 4 cells, but L-asp exposure did not result in any significant changes in HL-60 cells. On the other hand, L-asp pre-treatment did not affect topoisomerase-I (Topo-I) inhibitor, camptothecin (CPT)-induced DNA strand breaks or toxicity in Molt 4 cells. Our data imply that a decrease in S- and G2/M-phase cells following L-asp treatment may explain the reduction of etoposide-induced DNA lesions and cytotoxicity in Molt 4 cells, since topoisomerase-II (Topo-II) content or activity is a function of cellular proliferation status.     

The influence of Na+, K+-pump blockade on doxorubicin-mediated cytotoxicity and DNA strand breakage in human tumor cells

Summary We have previously shown that blockade of the Na⁺, K⁺-pump by the cardiac glycoside ouabain produces doxorubicin resistance and decreases topoisomerase II-mediated DNA strand breakage in hamster cells. To determine if this were a general phenomenon, the effect of pump blockade on doxorubicin resistance was assessed in three human tumor cell lines: A549 lung and HT29 colon adenocarcinomas and U1 melanoma. When cells were exposed to 1 μM ouabain prior to and during incubation with doxorubicin, cytotoxicity was markedly reduced. Ouabain had no effect on either the influx or the efflux of doxorubicin. However, all cell lines showed a ouabain-induced decrease in doxorubicin-induced topoisomerase-mediated DNA strand breakage (SSB). These data suggest that blockade of the Na⁺, K⁺ pump decreases doxorubicin cytotoxicity in human tumor cells by inhibiting topoisomerase-mediated SSB. Furthermore, they indicate that altered ionic gradients are a potential cause of resistance to drugs that use topoisomerase II as a target. This research was supported in part by a grant from the Rackham Foundation. Presented in part at the meeting of the American Association for Cancer Research in May, 1989     

Relationship between cytotoxicity and DNA strand breakage produced by adriamycin and other intercalating agents

Intercalating agents were compared with respect to cytotoxicity and DNA damage in order to further clarify the mechanism of action of this important group of anti-tumor drugs. Using the DNA alkaline elution technique, we have previously reported that when L1210 cells are exposed to a variety of intercalators, DNA single strand breaks are formed. The single strand breaks are spatially associated with DNA-protein crosslinks and the breaks and cross-links occur with approximately equal frequency. In this work, the cytotoxic potency of these drugs were compared using a soft agar colony forming assay. Ellipticine, which had produced the greatest frequency of single strand breaks, was only weakly toxic while adriamycin and actinomycin D exhibited potent cytotoxicity but produced fewer single strand breaks per unit drug concentration. To test the possibility that DNA repair processes may be involved in the cytotoxicity of intercalating agents, drug-treated cells were allowed to form colonies in the presence of caffeine, an inhibitor of DNA replication repair. Cytotoxicity of ellipticine, actinomycin D and hycanthone were significantly increased while that of adriamycin was diminished. We conclude that intercalating agents cause the formation of protein-associated DNA breaks but the role of these breaks in drug-induced cytotoxicity is unclear. Response to post-treatment incubation with caffeine suggests that the mechanism of cytotoxicity with adriamycin may differ in important respects from that of other intercalating agents. Extensions of these studies may be useful in elucidating the molecular basis for the biological interaction between radiation and intercalators.     

Synergistic cytotoxicity and DNA strand break formation by bromodeoxyuridine and bleomycin in human tumor cells

5-Bromo-2'-deoxyuridine (BrdUrd) is a thymidine analogue whose cellular effects are related to its incorporation into DNA. BrdUrd is a known radiosensitizing agent that could potentially enhance the activity of chemotherapeutic agents that interact directly with DNA. Therefore we studied the interaction of BrdUrd and bleomycin in a human head and neck squamous carcinoma cell line, SQ20B. Using a colony-forming assay and analyzing results by the median-effect method, we have shown that there is synergistic cytotoxicity between BrdUrd and bleomycin. Synergism is evident when BrdUrd is administered prior to bleomycin or when the two drugs are applied simultaneously and is evident at a variety of BrdUrd:bleomycin concentration ratios. Alkaline elution of DNA from cells exposed to BrdUrd and bleomycin demonstrated greater single strand break formation than expected from the individual single strand break frequencies induced by each drug alone. BrdUrd did not affect the rate of repair of bleomycin-induced single strand breaks or the formation of double strand breaks. Although the mechanism of this interaction at the molecular level is unclear, our studies suggest that a direct interaction of bleomycin with BrdUrd-substituted DNA may be the cause of the synergism of these two agents.     

Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein

Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, suppressed growth and induced differentiation in HL-205 cells, a clonal population of the human promyelocytic HL-60 leukemia cells, and in K-562-J cells, a clonal population of the human erythroid K-562 leukemia cells. Maturing HL-205 cells acquired either granulocytic or monocytic markers, namely, reactivity with the murine OKM1 monoclonal antibody, expression of nitroblue tetrazolium dye reduction, and staining for nonspecific esterase. The maturing K-562-J cells stained with benzidine, which indicates the presence of hemoglobin, an erythroid maturation marker. Although the acquisition of the maturation markers in both HL-205 and K-562-J cells was time dependent up to 6 days, the kinetics of this induction differed between the two cell types. Despite the in vitro inhibitory effect of genistein, treatment of either HL-205 or K-562-J cells with 150 micrograms/ml genistein for up to 16 h did not alter topoisomerase II activity (as determined by the unknotting assay) in their nuclear extracts. Analysis with the anti-phosphotyrosine PY-20 murine monoclonal antibody indicated that treatment of K-562-J cells with genistein decreased the reactivity of the antibody with two of the cellular proteins. However, no reactivity with the PY-20 antibody was detected in untreated or genistein-treated HL-205 cells. An early event in the HL-205 and K-562-J cells, occurring after only 1 h of treatment with 30-200 micrograms/ml genistein, was the induction of DNA damage as measured by an alkaline elution assay. This damage may be a contributing factor in the genistein-induced cell differentiation in the HL-205 and K-562-J cells.     

Elevation of c-myc protein by DNA strand breakage

The intracellular level of the c-myc protein is elevated in a dose and time dependent manner by DNA strand breakage. Lesions introduced by either alkylating agents or gamma irradiation are capable of stimulating increased production of c-myc protein. In addition, inhibition of DNA strand break rejoining maintains the level of the c-myc protein in an elevated state, whilst inhibition of DNA replication does not cause an increase in c-myc protein. We conclude that chromatin perturbation via DNA strand breakage both increases the endogenous amounts of the c-myc protein and maintains its elevated level.     

Thermal enhancement of DNA strand breakage in mammalian cells treated with bleomycin.

Chinese hamster ovary cells were treated with various doses of Bleomycin (BLM) for 1 hr at 37° or 43°C and the effects of these treatments in terms of cell killing and DNA strand breakage were determined. The technique of alkaline elution was utilized for the sensitive detection of the DNA strand breaks thus allowing the use of doses of BLM which also permit the measurement of cell survival. The results of these experiments showed that both the cytotoxic effects and DNA strand breakage were enhanced when the treatments were conducted at 43°C compared to 37°C. However, when the data were plotted as the degree of strand breakage vs the fraction of cells surviving, it appeared that the strand breaks produced under hypertbermic conditions were more effective for cell killing. While most of the breaks were rapidly repaired following treatment at 37°C, the rate of red was reduced and the amount of residual unrepalred breaks was greater when the drug treatment was at 43°C. Thus, it would appear that the repair of BLM induced DNA strand breaks is important for cell survival.     

Reduction by caffeine of adriamycin-induced cell killing and DNA damage in Chinese hamster cells: correlation with modulation in intracellular adriamycin content

The effect of caffeine, at concentrations between 10 microM and 20 mM was studied on Adriamycin-induced cytotoxicity and DNA damage in exponentially growing Chinese hamster V79 cells. Simultaneous administration of caffeine with 0.4 micrograms/ml (0.69 microM) Adriamycin for 1 h resulted in a concentration-dependent reduction in cell killing. The surviving fraction increased from 0.001 for cells treated with Adriamycin alone to 0.14 for cells treated in the presence of 1 mM caffeine and to 0.8 for cells treated at caffeine concentrations higher than 6 mM. A significant reduction in Adriamycin-induced cell killing was also caused by caffeine at micromolar concentrations where the surviving fraction increased from 0.00076 to 0.0014 (2-fold) after treatment with 10 microM, to 0.0038 (5-fold) after treatment with 20 microM and to 0.01 (13-fold) after treatment with 100 microM caffeine. Treatment of cells with caffeine for 1 h immediately after Adriamycin exposure (0.4 micrograms/ml, 1 h) resulted in a dose-dependent increase in survival as well, but the effect was smaller than that observed after simultaneous administration (increase in the surviving fraction from 0.003 to about 0.05 at concentrations higher than 5 mM). The reduction of Adriamycin-induced cytotoxicity by caffeine was reflected by a decrease in the slope of the survival curve, and it was similar over the entire range of Adriamycin and caffeine concentrations examined. The ability of cells to accumulate Adriamycin was reduced by caffeine from 43 ng/10(6) cells after treatment for 1 h in the presence of 0.5 micrograms/ml Adriamycin to 16 ng/10(6) cells for cells treated in the presence of 2 mM and to 8 ng/10(6) cells for cells treated in the presence of 10 mM caffeine. Induction by Adriamycin of DNA breaks, as assayed by the alkaline filter elution technique, was linear with concentration and was decreased in the presence of caffeine. The response to caffeine of Adriamycin-induced killing and DNA damage was similar, and it was only slightly different from the modulation induced in intracellular Adriamycin content. Compared to the effect of caffeine on cells exposed to ionizing radiations or other cytotoxic compounds, the results indicate an entirely different mode of caffeine action with anthracyclines. In addition, the results suggest caffeine-induced modulations in intracellular drug accumulation as an important determinant for the effect and may have useful implications in the clinical application of these compounds.     

DNA damage and cytotoxicity of mitoxantrone and doxorubicin in doxorubicin-sensitive and-resistant human colon carcinoma cells

Summary The effects of mitoxantrone (Mx) and doxorubicin (Dx) on cytotoxicity and DNA damage as assayed by alkaline elution were studied in two human colon adenocarcinoma cell lines sensitive (LoVo) and resistant (LoVo/Dx) to doxorubicin. Mx was more cytotoxic than Dx to LoVo cells and was partially cross-resistant in LoVo/Dx. In LoVo cells, Mx produced about 5 times more DNA single-strand breaks (DNA-SSB) than Dx, but both drugs caused an equal number of DNA double-strand breaks (DNA-DSB). In LoVo/Dx cells, the number of DNA-DSB was very low for both Dx and Mx, but DNA-SSB were about 20 times higher for Mx. In LoVo cells, the number of DNA-DSB and protein-associated SSB were similar at equitoxic concentrations. For LoVo/Dx, the partial cross-resistance of Mx might be explained by the much higher number of DNA-SSB produced by this drug.     

Nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells by inhibition of myeloperoxidase

Chronic inhalation of environmental particles is associated with pulmonary carcinogenesis. Although the mechanism has not yet been fully elucidated, influx of inflammatory cells, including neutrophils, is suggested to play a major role in this process. Typically, in the particle-exposed lung, influx of neutrophils is accompanied by an accumulation of nitrite. Previous studies indicated that nitrite may affect the toxicity of neutrophils, involving an interaction with neutrophil-derived myeloperoxidase (MPO). To evaluate the possible consequences of this interaction for inflammation-mediated genotoxicity, we investigated the effect of nitrite on neutrophil-induced DNA damage in pulmonary target cells. Therefore, activated neutrophils were co-cultured with alveolar type II epithelial cells (RLE), and DNA strand breakage was evaluated using single-cell gel electrophoresis (comet assay). In this system, addition of nitrite caused an increase in neutrophil-induced DNA strand breakage in RLE cells, which was associated with an inhibition of MPO activity. Similar results were obtained by co-culturing RLE cells with neutrophils in the presence of the specific MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). To further investigate the mechanism underlying these observations, in vitro experiments were performed using mixtures of nitrite, MPO and its substrate H2O2. DNA strand breakage by reagent H2O2 was inhibited when it was allowed to react with MPO before addition to the RLE cells. However, when MPO and H2O2 were pre-mixed in the presence of nitrite or 4-ABAH, the inhibitory effect of MPO on resultant DNA damage was reversed. Further studies using catalase indicated that DNA strand breakage by the pre-mixtures of MPO, H2O2 and nitrite was H2O2-specific, suggesting that nitrite prevents consumption of H2O2 by MPO. Collectively, our results show that nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells. This effect is probably due to an inhibition of MPO activity, which increases the availability of its DNA strand breaking substrate H2O2.     

Dependence of etoposide-induced cytotoxicity and topoisomerase II-mediated DNA strand breakage on the intracellular ionic environment

We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.     

DNA strand breakage and ADP-ribosyl transferase mediated DNA ligation during stimulation of human bone marrow cells by granulocyte-macrophage colony stimulating activity

ADP-ribosyl transferase (ADP-RT) is a chromatin-bound nuclear enzyme catalysing the transfer of ADP-ribose from NAD+ to chromatin proteins. The enzyme is activated by DNA strand breaks and has been suggested to have roles in both DNA repair (via its effect on DNA ligase II) and in differentiation. We recently demonstrated that specific inhibitors of ADP-RT preferentially inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage and that the specific proliferation/differentiation stimulus granulocyte-macrophage colony stimulating activity (GM-CSA) activates ADP-RT in human marrow cells within 3 h of exposure. The purpose of this study was to investigate the role of ADP-RT in monocyte-macrophage differentiation. By altering the time of addition of ADP-RT inhibitor it was demonstrated that maximal inhibition of macrophage differentiation only occurs when the inhibitor is added within the first 24 h of culture. This suggests that it is an early event during the induced differentiation of granulocyte-macrophage progenitor cells which requires ADP-RT. Fluorometric assay of the level of DNA strand breaks showed that GM-CSA induces DNA strand breaks which are rapidly ligated only if ADP-RT is available. These data and those of our earlier studies suggest that DNA rearrangement may be involved in differentiation of granulocyte-macrophage progenitors to the monocyte-macrophage pathway. Such a DNA rearrangement could provide a molecular basis for commitment of multipotent progenitors to a single lineage.     

Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster cells

Intercalator-induced DNA strand breaks in mammalian cells represent topoisomerase II:DNA complexes trapped by intercalators. These complexes are detected as protein-associated DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB) by filter elution. Using Chinese hamster lung fibroblasts (V79 cells) that were treated for 30 min with various concentrations of 4'-(9-acridinylamino)methanesulfon-m-anisidide or 5-iminodaunorubicin, we measured DNA strand breaks (SSB and DSB), sister chromatid exchanges (SCE), mutations at the hypoxanthine:guanine phosphoribosyltransferase locus, and cell killing. Further, we correlated DNA strand breakage with the three other parameters. Both drugs induced SCE, mutations, and cell killing at concentrations which also produced reversible DNA strand breaks. While the quantity of DSB correlated with SCE, mutations, and cytotoxicity for both drugs, we found more SCE, mutations, and cytotoxicity per SSB in cells treated with 5-iminodaunorubicin than in those treated with 4'-(9-acridinylamino)methanesulfon-m-anisidide. These data show that the DSB (but not the SSB) induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and 5-iminodaunorubicin at DNA topoisomerase II binding sites correlated closely with SCE, mutations, and cell killing and could therefore be responsible for their production.     

DNA polymerase beta mediates protection of mammalian cells against ganciclovir-induced cytotoxicity and DNA breakage

The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase beta (beta-pol) inhibitor, prunasin; (b) DNA beta-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and beta-pol-complemented cell lines; (c) methoxyamine, an inhibitor of beta-pol-dependent short-patch base excision repair, sensitized wild-type and complemented beta-pol cells to GCV, whereas it had no effect on the sensitivity of beta-pol-null cells to GCV. Because methoxyamine-mediated sensitization of beta-pol wild-type and beta-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both beta-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.     

Potentiation of bleomycin cytotoxicity in cultured mammalian cells by polyacrylic acid. III. Induction of DNA strand breakage and suppression of cellular heat production.

The 2E cytotoxicity of bleomycin toward cultured mammalian cells was synergistically enhanced by vortex-stirring in the presence of a low dose of high molecular weight polyacrylic acid. Cellular DNA isolated immediately after the above treatment suffered from severe single strand breaks. Heat production of the treated cells also decreased sharply immediately after the treatment, indicating that some functional disorder was probably induced on the cell membrane leading to cell death, possibly resulting from enhanced DNA strand breaks.     

Potentiation of doxorubicin cytotoxicity by buthionine sulfoximine in multidrug-resistant human breast tumor cells

Resistance to antineoplastic drugs is a major problem in the clinical management of cancer. Previous studies have demonstrated that the cytotoxicity of certain anticancer drugs is increased by lowering the glutathione (GSH) levels with buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. In this study we report that the resistance to doxorubicin, an anthracycline antibiotic and the most active agent in the treatment of breast cancer, can be partially reversed by exposing MCF-7 doxorubicin-resistant breast tumor cells (MCF-7/ADRR) to minimally cytotoxic doses of BSO. We found that the BSO treatment (50 microM, 48 h) of MCF-7/ADRR cells resulted in 80 to 90% depletion in total GSH concentrations. The toxicity of doxorubicin, as determined by growth inhibition and clonogenic assays, was significantly potentiated in the BSO-treated GSH-depleted cells relative to control breast tumor cells, and a dose-modifying factor of 5 to 7 was observed. Since the cytotoxicity of doxorubicin has been associated with its ability to undergo enzymatic activation and to form hydroxyl (OH) radicals in this cell line, we also quantitated the OH formation in the BSO-treated and untreated MCF-7/ADRR cells using electron spin resonance spintrapping techniques. These results show that doxorubicin stimulated at least 2-fold more OH formation in the tumor cells after GSH levels were decreased by 90%. These results indicate that GSH plays an important role in modulating doxorubicin-induced OH formation via the scavenging of hydrogen peroxide by glutathione peroxidase and thus partially protects MCF-7/ADRR cells from the cytotoxic effect of doxorubicin.     

Role of DNA breakage in cytotoxicity of doxorubicin, 9-deoxydoxorubicin, and 4-demethyl-6-deoxydoxorubicin in murine leukemia P388 cells

Formation and persistence of DNA single- and double-strand breaks stimulated by doxorubicin, 9-deoxydoxorubicin, or 4-demethyl-6-deoxydoxorubicin in murine leukemia P388 cells were compared in relation to drug DNA affinity, cellular pharmacokinetics, and cytotoxicity. Although cellular uptake and retention and DNA affinity of the anthracycline derivatives were similar to those of the parent drug, cytotoxic potency was quite different, 9-deoxydoxorubicin being much less cytotoxic than doxorubicin, and 4-demethyl-6-deoxydoxorubicin the most effective agent. After 1-h exposure of cells to cytotoxic drug levels, the extent of DNA strand breaks produced by 4-demethyl-6-deoxydoxorubicin was greater than that produced by doxorubicin, whereas 9-deoxydoxorubicin induced very few DNA breaks. As for the parent drug, proteolytic treatment of cell lysates on the filter was needed to detect DNA cleavage produced by the analogues. A linear increase of DNA breaks was observed for 2 h following 4-demethyl-6-deoxydoxorubicin or doxorubicin addition; by contrast, DNA break levels reached a plateau after 45 min of exposure to 9-deoxydoxorubicin. DNA lesions produced by the derivatives persisted, and doxorubicin-induced DNA breaks even increased after drug removal, indicating an absence of DNA break resealing under our conditions. These observations indicate that modifications of the chromophore moiety of the anthracycline may enhance both drug cytotoxicity and specificity of drug-target interactions, and thus provide further strong evidence that the anthracycline effect on DNA integrity is a critical aspect of the mechanism of drug action.     

DNA strand breakage and DNA adducts in lymphocytes of oral cancer patients

In lymphocytes of 12 oral cancer patients (and two control groups)the frequencies of DNA single-strand breaks and DNA-proteincross-linking were determined by alkaline filter elution. Wefound elevated DNA elution rates, which must be interpretedas an increased strand breakage frequency. There were significantcorrelations between the DNA strand breakage frequency and smokinghabits. Using the 32P-postlabelling assay we determined theDNA adduct level in lymphocytes of 23 oral cancer patients (andtwo control groups). No significant influence of smoking habiton the DNA adduct level could be detected. There was a significantcorrelation between the DNA adduct level and the     

Modulation of etoposide cytotoxicity and DNA strand scission in L1210 and 8226 cells by polyamines

The anticancer agent etoposide (VP-16) produces DNA strand scission in intact tumor cells or isolated nuclei. This activity may be mediated by topoisomerase II, an enzyme capable of producing double strand breaks in mammalian cells. Two established tumor cell lines were examined to see whether polyamines, which alter DNA conformation and topoisomerase II activities, affected the cytotoxicity, strand scission, and antitumor efficacy of VP-16. L1210 murine leukemia and 8226 human myeloma cells were treated with alpha-difluoromethylornithine (DFMO) to reduce intracellular polyamine levels via inhibition of ornithine decarboxylase. The polyamines putrescine and spermidine were markedly reduced by a 48-h incubation with 50 microM DFMO. This DFMO concentration did not inhibit colony formation in either cell line, but did reduce the growth rate of both cultures. In contrast, VP-16 produced a dose-dependent inhibition of colony formation. This was especially marked in the 8226 cell line. This correlated with DNA single strand breaks (SSBs) detected by the alkaline elution technique. When cells previously treated with DFMO were exposed to VP-16, a synergistic inhibition of colony formation (determined by isobologram analysis) was observed. However, VP-16-induced SSBs were only marginally increased by the DFMO pretreatment. When putrescine was combined concurrently with VP-16, both the in vitro cytotoxic effects and the number of DNA SSBs in L1210 cells were significantly reduced. These results demonstrate that putrescine inhibits VP-16-induced SSBs and commensurate cytotoxic effects, while DFMO, which depletes intracellular putrescine and partially reduces intracellular spermidine, acts to produce synergistic cytotoxic effects when combined with VP-16.