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W E Ross

Publications and source records attributed to W E Ross.

At least 37 records · Page 2Linked to original sources
PubMed1987

DNA topoisomerase II: a primer on the enzyme and its unique role as a multidrug target in cancer chemotherapy.

Based on the weight of evidence accrued in the past eight years, there is little question that the nuclear enzyme, topoisomerase II, serves as a common intracellular target for the cytotoxic effect of drugs of widely varying structure. The enzyme appears to be unique as a chemotherapy target in that it is recruited into a lethal process under the influence of drug. Its role contrasts sharply with other more classical chemotherapy targets, such as dihydrofolate reductase, whose activity must be successfully inhibited for the expression of cytotoxicity. Resistance to inhibitors of this enzyme frequently results from marked elevations in intracellular enzyme content. In contrast, the presence of topoisomerase is required for drug effect, and, in general, the greater the cellular content of the enzyme, the more sensitive the cell will be to these agents. However, important issues remain unresolved. The biochemical events that are initiated by cleavable complex formation and result in cell death must be more fully defined. It is likely a better understanding of the drug-enzyme interaction will be required for rational drug development. Finally, those aspects of the drug-topoisomerase interaction that confer therapeutic selectivity and/or clinical resistance are of paramount importance if the phenomenon is ever to be fully exploited.

Animals↗
PubMed1987

Topoisomerase-specific drug sensitivity in relation to cell cycle progression.

The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.

Animals↗
PubMed1987

Role of proliferation in determining sensitivity to topoisomerase II-active chemotherapy agents.

We have examined the relationship between topoisomerase II content and the DNA cleavage activity and cytotoxicity of etoposide during proliferative and quiescent culture conditions. In proliferating cultures of Chinese hamster ovary (CHO) cells, human lymphoblastic CCRF cells, and mouse leukemia L1210 cells, there was easily detectable topoisomerase II by immunoblotting. In contrast, quiescent CHO cells contained virtually no detectable topoisomerase II, while the content of L1210 cells was unchanged. Enzyme content of quiescent CCRF cells was diminished but detectable. DNA cleavage activity induced by etoposide correlated well with enzyme content in proliferating and quiescent cells. Quiescent CHO and CCRF cultures were highly resistant to the cytotoxic effects of etoposide as expected. However, despite unchanged enzyme content and DNA cleavage activity, there was also significant resistance observed in plateau L1210 cells. We have also investigated topoisomerase content and drug activity as a function of cell cycle progression. Following serum stimulation of confluent BalbC/3T3 cells, maximal etoposide-induced DNA cleavage activity is observed in G2/M and is associated with an increase in topoisomerase II content. Maximum cytotoxicity, however, occurs during mid to late S phase. Our data suggest that topoisomerase II content may be an important determinant of chemotherapeutic sensitivity during alterations in the proliferative status of the cell. However, it is clear that other factors must be involved in cell sensitivity, and elucidation of these may contribute to our understanding of the mechanism of action of these drugs.

Animals↗
PubMed1987

Mediation of multi-drug resistance in a Chinese hamster ovary cell line by a mutant type II topoisomerase.

Identification of DNA topoisomerase II as the intracellular target for the DNA cleavage activity of the epipodophyllotoxins and several intercalating agents is well established. In contrast, definite correlation of cleavable complex formation with eventual cell death has been more difficult to document. Our studies with an epipodophyllotoxin resistant cell line not only provide additional evidence that the enzyme is a multi-drug target, but also implicate drug-stimulated cleavage activity as a critical component of cytotoxic effect. When compared to WT (wild type) cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activity of etoposide as well as various intercalating agents. Steady state concentrations of these drugs in both cell lines is identical. Enzyme content as measured by immunoblot and by total decatenation activity in crude nuclear extracts is equal. Catalytic activity is also equally sensitive to inhibition by etoposide. In contrast, cleavage activity in purified enzyme from VpmR-5 cells is profoundly resistant to stimulation by drug. These data indicate that a multi-drug resistant phenotype may be acquired by a qualitative change in the enzyme that alters its interaction with drug. Further, the data strongly support a direct role for cleavable complex formation in cell death.

Amsacrine↗
PubMed1987

DNA topoisomerases in cancer therapy.

DNA topoisomerases I and II are nuclear enzymes which modify DNA topology by their ability to break and reseal one or both strands in concert. Each of these enzymes has important functions in DNA replication, and very likely in other genetic processes as well. In addition, each can serve as a cancer chemotherapy target. The plant alkaloid camptothecin traps DNA topoisomerase I in a form which is covalently linked to DNA. Presumably this action is the basis for its anti-tumor effect, although this has not been conclusively demonstrated. The evidence for DNA topoisomerase II as a target for intercalating agents and epipodophyllotoxins is more formidable. Each of these classes of agents traps the enzyme on DNA in a structure referred to as a 'cleavable complex'. Illicit recombination events, as well as induction of an 'SOS-like' response analogous to that found in bacteria, have been suggested as possible mechanisms for cell death following cleavable complex formation. Development of new agents directed at topoisomerase II will depend heavily on understanding the nature of the interaction between the drug, enzyme and DNA. Hypotheses are presented in this paper on this interaction. Intracellular content of topoisomerase II is linked to the ability of the cell to enter a G0-like state, and the inability of malignant cells to undergo such a change may provide part of the basis for therapeutic selectivity. The ability to modulate topoisomerase II levels may provide an opportunity for therapeutic intervention.

Antineoplastic Agents↗
PubMed1986

Proliferation dependence of topoisomerase II mediated drug action.

Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.

Aminoacridines↗
PubMed1985

Effects of verapamil on etoposide, vincristine, and adriamycin activity in normal human bone marrow granulocyte-macrophage progenitors and in human K562 leukemia cells in vitro.

We have examined the effects of verapamil on the cytotoxicity of etoposide, vincristine, and Adriamycin in human leukemia K562 cells as well as in normal human bone marrow granulocyte-macrophage progenitors (CFU-GM). Etoposide was 10-fold more potent against K562 cells than against normal human bone marrow CFU-GM. Similarly, vincristine cytotoxicity was about 10-fold greater against K562 cells than against human bone marrow CFU-GM. In contrast, Adriamycin exhibited little selectivity for K562 cells versus normal bone marrow CFU-GM during the 1-h incubation period of the experiments. In the presence of verapamil (2.5-10 microM), etoposide cytotoxicity was enhanced in both malignant and normal cells. Verapamil enhanced vincristine (0.1 microM) cytotoxicity in K562 cells but did not potentiate Vinca alkaloid toxicity in normal bone marrow CFU-GM. Adriamycin, on the other hand, did not display any calcium antagonist-mediated potentiation of cytotoxicity in either malignant or normal tissue. These results indicate that short-term (1 h) incubations with etoposide, vincristine, and Adriamycin yield different profiles of toxicities whether used alone or with chemosensitizing agents such as the calcium antagonists. These differences in activities are consistent with different mechanisms for intracellular disposition of these three classes of anticancer agents and are worthy of further investigation, especially with regard to combinations with calcium antagonists.

Bone Marrow↗
PubMed1985

A method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin.

This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [32P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion.

Animals↗
PubMed1985

Verapamil-induced augmentation of etoposide accumulation in L1210 cells in vitro.

The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (etoposide) (VP-16) as well as on subsequent DNA damage and cytotoxicity were studied in L1210 cells in vitro. At extracellular VP-16 concentrations of 1 to 5 microM, verapamil (10 microM) addition resulted in an increase of DNA single-strand break frequency comparable to that found when VP-16 was present alone at a 3-fold higher concentration. In addition, the elevation of cellular VP-16 levels in the presence of verapamil was linearly correlated with the enhancement of DNA damage and increased cell kill. Verapamil-mediated increase in net VP-16 transport was rapid (1 to 2 min), and allowed for the same elevation of steady-state VP-16 concentration, whether verapamil was added simultaneously with VP-16 or was added after a steady-state level of VP-16 was achieved. Verapamil-mediated elevation of VP-16 levels was not seen at reduced temperature (0 degrees C). Studies of bidirectional VP-16 transport revealed that verapamil (40 microM) did not alter influx of VP-16 (15 microM), but lowered the unidirectional rate constant for efflux by 93%, resulting in the observed increase of steady-state level of the epipodophyllotoxin. Removal of verapamil resulted in a rapid return of VP-16 to levels comparable to that seen with VP-16 alone. When VP-16 was allowed to flow out of the cell in the presence of verapamil, less than 5% of cellular epipodophyllotoxin was retained, suggesting that the calcium antagonist is not acting by enhancing intracellular binding of VP-16. These results indicate that verapamil potentiates VP-16 activity by elevation of intracellular exchangeable epipodophyllotoxin; an activity which seems to be due to inhibition of the efflux mechanism for VP-16. The low intracellular retention of this epipodophyllotoxin and the good correlation between intracellular VP-16 and subsequent DNA damage and cytotoxicity suggest that the epipodophyllotoxin class of anticancer agents may be more useful for probing calcium antagonist effects on drug transport in sensitive cells and in cells exhibiting pleiotropic drug resistance than the vinca alkaloids and anthracyclines which have large tight binding intracellular components.

Animals↗
PubMed1984

Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells.

Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05-1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.

Adenosine Triphosphate↗
PubMed1984

Inhibition of etoposide-induced DNA damage and cytotoxicity in L1210 cells by dehydrogenase inhibitors and other agents.

The mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), an important antitumor agent, is unclear. There is evidence that DNA may be the target of action because VP-16 causes single-strand and double-strand breaks in DNA and produces cytotoxicity over a similar dose range. We have hypothesized that an enzyme system, such as dehydrogenase, catalyzes an oxidation-reduction reaction involving the pendant phenolic group which forms an active metabolite that causes the DNA damage and cytotoxicity. To test our hypothesis, we investigated the effect of disulfiram, an aldehyde dehydrogenase inhibitor, and its metabolite, diethyldithiocarbamate, on VP-16-induced DNA damage in L1210 cells. Using the alkaline elution technique to assay DNA damage, we found that disulfiram and diethyldithiocerbamate inhibited VP-16-induced single-strand breaks. Both compounds were also capable of significantly reducing VP-16-induced cytotoxicity. Oxalic acid, pyrophosphate, and malonic acid, competitive inhibitors of succinate dehydrogenase, and the naturally occurring dehydrogenase substrates, succinic acid, beta-glycerophosphate, and isocitric acid, also blocked the effects of VP-16. Free-radical scavengers were also studied. While sodium benzoate was particularly effective in preventing drug-induced DNA damage and cytotoxicity, a number of other scavengers were not. Our data are consistent with the hypothesis that VP-16 is activated by an enzyme such as a dehydrogenase which transforms it into an active intermediate resulting in DNA damage and, consequently, cell death.

Aldehyde Dehydrogenase↗
PubMed1984

Probenecid inhibition of methotrexate cytotoxicity in mouse L1210 leukemia cells.

The effect of probenecid on methotrexate cytotoxicity has been measured using mouse L1210 leukemia cells in vitro. Cytotoxic effects were measured using a soft agar cloning technique. Cell exposure to varying concentrations of methotrexate at fixed concentrations of probenecid resulted in increased cell survival when compared with exposure to methotrexate alone. Cell exposure to a fixed concentration of methotrexate and varying concentrations of probenecid showed this effect to be dependent on the concentration of probenecid. Intracellular methotrexate concentrations were unchanged by the presence of probenecid. However, there was an effect of probenecid on progression of cells through the cell cycle which would tend to decrease the number of cells susceptible to the cytotoxic effects of methotrexate.

Animals↗
PubMed1984

Potentiation of etoposide-induced DNA damage by calcium antagonists in L1210 cells in vitro.

Verapamil and a number of other Ca2+ antagonists were found to potentiate DNA damage induced by 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside (VP-16) in L1210 cells in vitro. The potentiating effect of verapamil on DNA single-strand breaks in vitro was concentration dependent, relevant to clinically achieved levels of Ca2+ antagonists, and showed good correlation with enhanced cytotoxicity when VP-16 and Ca2+ antagonists were combined in soft agar colony-forming assays. Onset of verapamil activity was observed within 20 min of addition to cells whether VP-16 had been preincubated with cells or was added simultaneously with the Ca2+ blocker. The presence of the extracellular Ca2+ antagonist was required for potentiation as evidenced by the rapid reversal of increased DNA single-strand breaks when cells were washed free of verapamil. Neither ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid nor the Ca2+ ionophore A23187 altered verapamil potentiation of VP-16-induced DNA damage, suggesting that this Ca2+ antagonist acts by a mechanism other than by inhibition of Ca2+ influx. In isolated L1210 nuclei, verapamil did not enhance VP-16- or 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-beta-D- glucopyranoside (VM-26)-induced single-strand breaks suggesting a requirement for the intact cell. Even though VM-26 was 5- to 10-fold more potent than VP-16, verapamil potentiated the DNA damage caused by these two epipodophyllotoxins in L1210 cells to the same extent when these agents were used at equipotent doses. Potency differences between VM-26 and VP-16 were evident in isolated nuclei suggesting that nuclear binding or activation is a more important parameter than were previously reported membrane transport differences. The significance of Ca2+ antagonist potentiation of VP-16-induced DNA damage is discussed in terms of overcoming resistance to epipodophyllotoxins and characterizing more precisely the intracellular disposition, binding, and activation of VP-16.

Animals↗
PubMed1983

Properties of a purified nuclear topoisomerase from L1210 cells.

A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.

Animals↗