Proliferation-dependent topoisomerase II content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells

We have shown previously that quiescent Chinese hamster ovary (CHO) cells are less sensitive than log phase CHO cells to the cytotoxic and DNA cleavage effects of etoposide, a drug which appears to act via DNA topoisomerase II. This loss of sensitivity was associated with a decrease in topoisomerase enzyme activity in nuclear extracts of the quiescent cells. We have now extended our observations by examining the basis for the reduction in enzyme activity during quiescence. DNA topoisomerase II content, as assayed by immunoblotting with a polyclonal rabbit anti-topoisomerase II antiserum, was virtually absent in nuclear extracts of quiescent CHO cells in contrast to logarithmically growing cells. This suggests that the previously demonstrated loss of enzyme activity in CHO cells is a function of reduction in content rather than posttranslational modifications of the enzyme. Quiescent human lymphoblastic CCRF cells also exhibited reduced topoisomerase II content compared to actively proliferating cultures, but the difference was less than that observed in CHO cells. In contrast, log and plateau phase cultures of mouse leukemia L1210 cells exhibited similar topoisomerase II content. Reduction in enzyme content correlated with the ability of these cell lines to accumulate during quiescence with a G0-G1 content of DNA. Sensitivity to the DNA cleavage effects of etoposide in dividing and nondividing cells correlated well with enzyme content. As has been observed with CHO cells, both CCRF and L1210 cells in plateau phase were more resistant to the cytotoxic effects of etoposide than those actively dividing. The result with L1210 cells was surprising, however, in light of the equivalent DNA damage observed under the two growth conditions. Our data indicate that topoisomerase II enzyme content is proliferation dependent in some but not all cells and suggest that while enzyme content may be important in drug resistance in some cell types, other factors can decrease the sensitivity of the cell to cleavable complex formation as well.     

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.     

Effect of cell proliferation and chromatin conformation on intercalator-induced, protein-associated DNA cleavage in human brain tumor cells and human fibroblasts

Antineoplastic intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilize a cleavable complex between topoisomerase II and DNA. The production of protein-associated DNA cleavage in whole cells exposed to m-AMSA is thought to represent the cellular correlate of this topoisomerase II-mediated reaction. Protein-associated DNA cleavage can be quantified in mammalian cells by using alkaline elution technology. In an attempt to understand the impact of phenotypic and biochemical cellular characteristics on protein-associated DNA cleavage, we quantified m-AMSA-induced DNA cleavage in quiescent or proliferative normal human fibroblasts (cell strain 1508) and human glioblastoma cells (line T98G) as well as in asynchronously proliferating HeLa cells. The magnitude of DNA cleavage in quiescent fibroblasts and quiescent glioblastoma cells was identical and low relative to that observed in the HeLa cells. The magnitude of DNA cleavage was enhanced in both cell types following proliferation. This enhancement was greater in the glioblastoma cells than in the fibroblasts. These results were not due to alterations in cellular m-AMSA uptake. Chromatin was more elongated (open) in the quiescent glioblastoma cells than in the quiescent fibroblasts (as visualized by using the premature chromosome condensation assay), suggesting chromatin accessibility to drug per se may not be a critical determinant of the magnitude of m-AMSA-induced DNA cleavage. The onset of the enhanced m-AMSA-induced DNA cleavability that accompanied proliferation closely followed the formation of regions of localized chromatin decondensation, a late G1 event, and coincided with the onset of enhanced thymidine uptake, a marker for the onset of S phase. m-AMSA-induced cytotoxicity was also enhanced in proliferating compared with quiescent cells. The major finding of this study is that the cellular target for m-AMSA, putatively topoisomerase II, is more susceptible to drug action in proliferating cells than in quiescent cells. Effects of chromatin conformation or cellular phenotype upon topoisomerase II-mediated events such as m-AMSA-induced DNA cleavage are less certain.     

Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells

The DNA intercalator, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase II-mediated protein-linked DNA strand breaks. This function of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia L1210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells and then treated with drug for 30 min. DNA modifications were assayed by alkaline elution. We found that the frequencies of m-AMSA- or VP-16-induced DNA-protein links were higher in nuclei from exponentially growing than in those from quiescent cells in both the 3T3 and the L1210 lines. Drug-induced protein-associated DNA breaks were also studied as a function of the cell cycle in 3T3 cells that had been arrested by contact inhibition in medium containing 1% calf serum and then stimulated to proliferate by raplating at a lower cell density in medium containing 10% serum. In these synchronized cells, a large peak of [3H]thymidine incorporation occurred 18-30 h after replating. The yield of DNA-protein cross-links produced by 30-min drug treatments of nuclei isolated at various times after growth initiation increased concomitantly with the peak of the DNA synthesis. The topoisomerase II activity of nuclear extracts, as measured by kinetoplast DNA decatenation followed a similar pattern. Using colony-forming assays, we also observed that m-AMSA and VP-16 were most cytotoxic in proliferative cells and during DNA synthesis. These results suggest that alkaline elution measurement of m-AMSA- or VP-16-induced protein-linked DNA breaks reflects the association of topoisomerase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time.     

Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro

The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy.     

Inhibition of Topoisomerase II by a Novel Antitumor Cyclic Depsipeptide, BE-22179

BE-22179, a novel cyclic depsipeptide antibiotic having two 3-hydroxyquinoline moieties, inhibited the DNA-relaxing activity of L1210 topoisomerase II completely at 0.08 μM . This effect was far stronger than that of VP-16. However, it did not show any marked effect on topoisomerase II-mediated DNA cleavage. BE-22179 was ineffective in inhibiting the DNA relaxation by topoisomerase I at concentrations up to 10 μM , but showed DNA-intercalating ability (DNA unwinding) at 30 μM . The structure of BE-22179 is quite novel for a topoisomerase II inhibitor. Echinomycin, a quinoxaline antibiotic structurally related to BE-22179, interfered with DNA relaxation by topoisomerase II, though the effect was not due to inhibition of the catalytic activity of topoisomerase II but to conformational change of DNA based on its intercalation into DNA. Therefore, the potent inhibitory activity on topoisomerase II might not be a common activity of quinoxaline antibiotics, but might rather be specific to BE-22179. BE-22179 prevented DNA synthesis as well as RNA synthesis in L1210 cells and inhibited the growth of the cells. However, it remains unclear to what extent the topoisomerase II inhibition was responsible for the cytotoxicity of BE-22179.     

Differential effects of amsacrine and epipodophyllotoxins on topoisomerase II cleavage in the human c-myc protooncogene.

Amsacrine and demethylepipodophyllotoxins (etoposide and teniposide) are potent topoisomerase II inhibitors which have optimum activity in different cancers. To investigate whether these differences are due to different activity on cellular oncogenes, drug-induced topoisomerase II cleavage sites were mapped and sequenced in the human c-myc protooncogene. In the presence of purified murine L1210 topoisomerase II, amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds to the entire promoter region (approximately 20 base pairs on the sides of the P2 site). In the case of teniposide or etoposide, cleavage is more diffuse and markedly less at the P2 site. Mapping of cleavage sites in human small cell lung carcinoma cells (NCI N417) also shows that cleavage in the P2 promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.     

Protein-linked DNA strand breaks produced by etoposide and teniposide in mouse L1210 and human VA-13 and HT-29 cell lines: relationship to cytotoxicity

The two demethylepipodophyllotoxin glycosides, teniposide (VM-26) and etoposide (VP-16), have previously been reported to interact with DNA topoisomerase II by stabilizing a topoisomerase II-DNA covalent intermediate. This study examined the protein-associated aspect of the topoisomerase II-DNA-epipodophyllotoxin lesion. We found that in mouse (L1210) and human (VA-13 and HT-29) log-phase cell cultures, all DNA strand breaks produced by VP-16 or VM-26 were protein-associated. We found also that these protein-associated breaks occurred with a frequency which correlated with cytotoxicity in all three cell lines. For all three cell lines and for both compounds the regression lines were similar. Therefore, for a given class of topoisomerase II inhibitors, it may be possible to generate a characteristic line from which DNA-protein crosslink frequency predicts cytotoxicity.     

Etoposide-induced DNA cleavage in human leukemia cells

Summary The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents such as etoposide, teniposide, and a variety of intercalating agents. These compounds cause the enzyme to cleave DNA, forming a DNA-protein complex that may be a key step leading to cell death. It is apparently unique as a chemotherapy target, since drug potency diminishes with decreasing enzyme activity. It was thus of interest to examine the topoisomerase content and drug-induced DNA cleavage in freshly obtained human leukemia cells and to compare the obtained data with the results of similar studies performed in well-characterized human leukemia cell lines. The human T-lymphoblast line, CCRF-CEM, was more than 100-fold more sensitive to the DNA-cleavage effect of etoposide than the cells of the 13 leukemic patients examined. One of the leukemia lines (HL-60) and a lymphoblastoid line (RPMI-7666) were somewhat less sensitive than cells of the CCRF-CEM cells, but were still 10-fold more sensitive than the patients studied. The relative insensitivity of the freshly obtained cells could not be accounted for by differences with respect to drug uptake but were associated with markedly reduced topoisomerase-II content as assayed by immunoblotting using a mouse polyclonal serum against topoisomerase II. Heterogeneity was observed in the sensitivities of patients' cells with respect to both drug-induced DNA cleavage and enzyme content. The observed differences between cultured cell lines and patients' cells may have been related to their proliferative status. Etoposide potency in normal resting lymphocytes resembles that observed in circulating leukemia cells. However, following mitogenesis with phytohemagglutinin and interleukin-2, proliferating lymphocytes become as sensitive to etoposide as cultured cell lines with regard to DNA cleavage. This effect was accompanied by an increase in topoisomerase-II content. Our data thus support the hypothesis that topoisomerase-II content may be an important determinant of cell sensitivity to certain classes of chemotherapy agents. Efforts to stimulate topoisomerase-II content may improve the therapeutic efficacy of these drugs.This work was supported by a USPHS grant (CA 40884)     

Calmodulin inhibitor trifluoperazine in combination with doxorubicin induces the selection of tumour cells with the multidrug resistant phenotype.

Trifluoperazine (TFP) is effective in modulating DNA damage/repair in doxorubicin (DOX) treated cells. In the present study we have characterised the resistance phenotype of parental sensitive L1210 mouse leukaemia cells (L1210/S) adapted to grow in the presence of 0.017 microns DOX+5 microM TFP (L1210/DT). Although with prolonged exposure, 0.017 microM DOX alone produced < 35% cell kill in L1210/S cells, similar cytotoxicity was achieved at 0.43 microM DOX in L1210/S cells selected in the presence of 0.017 microM DOX+5 microM TFP. L1210/DT cells were > 30-fold resistant to DOX following a 3 h drug exposure in a soft agar colony assay. In contrast, DOX sensitivity in cells adapted to grow in 5 microM TFP alone was comparable to L1210/S cells. Resistance to other inhibitors of topoisomerase II in L1210/DT cells was > 30-fold to etoposide and > 6-fold to amsacrine. The levels of the 170 kDa and 180 kDa isoforms of topoisomerase II in an immunoblot were comparable between the L1210/S and L1210/DT cells. Cross resistance to vincristine in the L1210/DT cells was accompanied by the overexpression of plasma membrane P-glycoprotein. Although a 1.5-2-fold decrease in accumulation of etoposide and DOX was observed in the L1210/DT cells, drug levels for equivalent DNA damage in the alkaline elution assay were > 5-fold higher in the L1210/DT versus L1210/S cells. No abrogation in the modulating effects of TFP on DOX, VP-16 or amsacrine induced cytotoxicity was apparent in the L1210/DT cells. Results suggest that: (a) TFP in combination with low concentrations DOX can induce the selection of cells with the multidrug resistant phenotype; and (b) characteristics of cells selected for resistance to DOX or DOX plus TFP are comparable.     

Effects of the bifunctional antitumor intercalator ditercalinium on DNA in mouse leukemia L1210 cells and DNA topoisomerase II

Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.     

Progressive resistance to doxorubicin in mouse leukemia L1210 cells with multidrug resistance phenotype: reductions in drug-induced topoisomerase II-mediated DNA cleavage

Cells selected for resistance to doxorubicin (DOX) express the multidrug resistance (MDR) phenotype, and resistance has been suggested to be due primarily to enhanced cellular efflux of drug. A progressively DOX-resistant (10- and 40-fold) L1210 mouse leukemia model system, which does not exhibit enhanced DOX efflux as a primary mechanism of resistance, was found to display the MDR phenotype, based on overexpression of P-glycoprotein in western blots and cross-resistance to vinca alkaloids. Cross-resistance to another topoisomerase II inhibitor, etoposide (VP-16), was similar to that of DOX (10- and 40-fold), whereas resistance to N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide (m-AMSA) was 5-fold lower. In contrast, no cross-resistance to camptothecin, an inhibitor of topoisomerase I, was observed. Topoisomerase II decatenation activity in nuclear extracts from 10- and 40-fold DOX-resistant cells was 2- and 4-fold lower, respectively, when compared to sensitive cells. In these cells, however, marked reductions in m-AMSA- and VP-16-induced topoisomerase II mediated DNA cleavage were found to exceed decreases in the catalytic activity of the enzyme. Results from this study demonstrated that, in progressively DOX-resistant L1210 mouse leukemia cells with the MDR phenotype, a better relation existed between the degree of resistance and reduced VP-16- and m-AMSA-induced topoisomerase II mediated DNA cleavage, than between increases in P-glycoprotein and concomitant reduction in DOX accumulation.     

DNA topoisomerase II as a target of antineoplastic drug therapy

Summary A major goal of cancer therapy research is identification of critical biochemical targets that mediate the ability of effective cancer chemotherapy to kill tumor cells while allowing the maintenance of normal cell function. A candidate for such a target is DNA topoisomerase II, a ubiquitous enzyme that alters three-dimensional conformation of supercoiled DNA. DNA intercalating agents and epipodophyllotoxins stabilize a DNA and topoisomerase II complex. The process of stabilization probably represents the poisoning of an intermediate state in the normal functioning of the enzyme. This stabilized intermediate state can be measured in whole cells using the filter elution method of Kohn to quantify protein-associated DNA cleavage produced when the cells are exposed to intercalators or epipodophyllotoxins. By altering cell populations in quantifiable ways, four factors appear to influence the magnitude of drug-induced, topoisomerase II-mediated DNA cleavage and cytotoxicity: (a) the proliferative state of the cell (proliferating cells are more sensitive than quiescent ones); (b) the cell cycle state (cells pharmacologically recruited into G₁-S are more sensitive than asynchronously growing cells); (c) the chromatin conformation (DNA methylation, polyamine depletion, and other chromosomal changes can alter the magnitude of topoisomerase II-mediated effects); (d) the cellular phenotype (in an as yet uncharacterized manner, malignant cells apparently are more sensitive to topoisomerase II-mediated events than normal cells). These data suggest that the biochemical basis of the therapeutic index of drugs such as the intercalating agents or epipodophyllotoxins may be the intrinsic hypersensitivity of the topoisomerase II in malignant cells to poisoning by these drugs.     

Intercalator-induced, topoisomerase II-mediated DNA cleavage and its modification by antineoplastic antimetabolites

Defining specific biochemical targets of active antineoplastic agents could aid in discovering better anticancer therapy and more thoroughly understanding the biochemical basis of malignancy. Through a series of cellular and biochemical studies, we and others have identified the nuclear enzyme topoisomerase II as the target of several active agents, including 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The interference with topoisomerase II produced by m-AMSA can be quantified in whole cells exposed to m-AMSA by using the alkaline elution technique to measure DNA cleavage. Antimetabolites such as ara-C, hydroxyurea, and 5-azacytidine can augment m-AMSA-induced, topoisomerase II-mediated DNA cleavage and, concurrently, m-AMSA-induced cell killing. Studies in proliferating and quiescent human cells and an m-AMSA-sensitive/resistant human leukemia cell pair further support the hypothesis that a connection exists between topoisomerase II-mediated DNA cleavage and the mechanism by which m-AMSA kills cells. Pharmacologic or hormonal modification of specific biochemical processes critical to drug-induced cytotoxicity may enhance the therapeutic index of clinically useful agents.     

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues

20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.     

Structure-activity relations,cytotoxicity and topoisomerase II dependent cleavage induced by pendulum ring analogues of etoposide

The cytotoxicity of etoposide and its analogues, dihydroxy (DHVP), o-quinone (VP-Q) and o-methyl (VP-OMe), was evaluated in human breast (MCF-7) and HL60 tumour cells. Although less potent than etoposide, both DHVP and VP-Q were cytotoxic to these cells. However, VP-OMe was inactive. Studies with purified topoisomerase II showed that the intensity of DNA cleavage and the pattern of cleavage were similar for DHVP, VP-Q and etoposide. In contrast, the VP-OMe failed to induce DNA cleavage, indicating that the presence of 4′-OH is essential for metabolism, induction of topoisomerase II-mediated DNA cleavage and cytotoxicity of etoposide and its analogues.     

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.     

Differential ability of 2,4-dinitrophenol to modulate etoposide cytotoxicity in mammalian tumor cell lines associated with inhibition of macromolecular synthesis

The inhibitor of oxidative phosphorylation, 2,4-dinitrophenol (DNP), abrogates etoposide cytotoxicity in murine leukemia L1210 cells without affecting the quantity of drug-induced DNA lesions. Cell cycle arrest and events associated with cell death were followed in etoposide treated L1210 cells under conditions in which DNP reduced cytotoxicity greater than 100-fold. Micronucleation, associated with mitotic catastrophe, and apoptotic internucleosomal degradation of DNA, were both inhibited by DNP co-treatment to an extent consistent with clonogenic survival. However, the ability of etoposide to cause cell cycle arrest was minimally affected by DNP. For the same proportion of cells arresting in G(2), DNP co-treatment profoundly reduced etoposide cytotoxicity, suggesting a separation between etoposide-induced G(2) arrest and cell death. At the same concentration used to treat L1210 cells, DNP was unable to abrogate etoposide cytotoxicity in HeLa, Chinese hamster ovary or HL60 cells. The relationship between ongoing macromolecular synthesis during etoposide treatment and clonogenic survival was further studied in L1210 and HeLa cells. In general, L1210 cells were more sensitive than HeLa cells to inhibition of macromolecular synthesis by DNP. A higher DNP concentration did partially block etoposide cytotoxicity in HeLa cells, in association with increased inhibition of macromolecular synthesis. It was not possible to attribute the reduced cytotoxicity of etoposide in HeLa cells to inhibition of DNA or RNA synthesis alone, because inhibitors with greater specificity (aphidicolin and DRB) had no effect on clonogenic survival. However, aphidicolin partially abrogated etoposide cytotoxicity in L1210 cells, although to a lesser extent than DNP. These data indicate that inhibition of DNA or RNA synthesis alone during etoposide exposure is insufficient to abrogate killing of HeLa cells, that inhibition of etoposide cytotoxicity in HeLa cells may require the additional inhibition of protein synthesis, and that the modulating effects of ongoing DNA synthesis on etoposide cytotoxicity are cell line dependent.     

Effect of retinoic acid on DNA cleavage and cytotoxicity of topoisomerase II-reactive drugs in a human head and neck squamous carcinoma cell line

Evidence from several in vitro systems indicates that cellular responses to DNA topoisomerase II-reactive compounds (i.e., the epipodophyllotoxins and intercalating agents) may be affected by the relative rate of proliferation. Using a human head and neck squamous carcinoma cell line 183A, we have investigated the effect of beta-all-trans-retinoic acid (RA), a substance with known antiproliferative effects, on the DNA cleavage and cytotoxic activities of etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide which interact with topoisomerase II. The effect of RA treatment on the activity of X-radiation and bleomycin, both of which produce free radical mediated effects, was also examined. RA treatment (10 to 20 microM for 72 h) does not significantly influence DNA cleavage induced by X-radiation or bleomycin but decreases DNA cleavage and cytotoxicity mediated by etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide. Further, this effect can be demonstrated at a dose of RA that is minimally growth inhibitory. The inhibitory effect of RA appears to be localized to the nucleus given that similar effects on drug-mediated DNA cleavage can be demonstrated in nuclei isolated from RA-treated cells. However, both drug-stimulated DNA cleavage activity and topoisomerase II catalytic activity are approximately equal in crude nuclear extracts of untreated and RA-treated cells. These data suggest that the resistance to topoisomerase II-reactive drugs induced by RA treatment of 183A cells is not mediated through a direct effect on the enzyme, but, instead, is related to other changes in the nuclear milieu occurring in the initial stages of quiescence such as altered chromatin conformation.     

Estrogen-induced potentiation of DNA damage and cytotoxicity in human breast cancer cells treated with topoisomerase II-interactive antitumor drugs

Hormone stimulation of responsive neoplasms is a potential strategy for improving the target selectivity of cancer chemotherapy. Using an alkaline DNA-unwinding technique to detect drug-induced DNA strand breakage, we have shown that estrogen stimulation of T-47D human breast cancer cells enhances induction of DNA cleavage by etoposide (VP-16), 4',9-acridinylaminomethanesulfon-m-anisidide (m-AMSA), mitoxantrone, and doxorubicin, drugs known to interact with the DNA-modifying enzyme topoisomerase II. No enhancement of DNA cleavage or cytotoxicity was seen in estrogen-treated cells exposed to X-rays or bleomycin. Novobiocin (an inhibitor of topoisomerase II) markedly antagonized the enhancing effect of estrogen on VP-16-induced DNA cleavage, while neutral nucleoid sedimentation detected less than 10% of such strand breaks revealed in estrogen-treated cells by alkaline unwinding. Estrogen did not affect DNA repair of lesions induced by X-rays, VP-16, or ultraviolet radiation. Enhancement of DNA cleavage was accompanied by a corresponding enhancement of cytotoxicity in cells treated with VP-16 or m-AMSA, but only minimal enhancement of cytotoxicity was seen following treatment with mitoxantrone or doxorubicin. Estrogen-treated and control cells treated with VP-16 and m-AMSA sustained similar levels of DNA cleavage for equivalent levels of cytotoxicity. These findings suggest that estrogen potentiates the cytotoxicity of VP-16 and m-AMSA by enhancing topoisomerase II-mediated DNA damage but that such "damage" does not contribute significantly to cytotoxicity induced by mitoxantrone or doxorubicin. Estrogen stimulation of receptor-positive breast cancer may prove to be a clinically relevant strategy for improving the selectivity and cytotoxicity of some, but not all, topoisomerase II-interactive drugs.