Janice Mayes

Circumvention of resistance by doxorubicin, but not by idarubicin, in a human leukemia cell line containing an intercalator-resistant form of topoisomerase II: evidence for a non-topoisomerase II-mediated mechanism of doxorubicin cytotoxicity

The novel, topoisomerase II-reactive anthracycline intercalator idarubicin (IDA) was demonstrated to produce protein-associated DNA cleavage in HL-60 human leukemia cells. Like a host of other antineoplastic intercalating agents, IDA produced this effect to a much lesser extent in HL-60/AMSA cells, a line that is primarily resistant to the intercalator amsacrine, but is cross-resistant to a variety of topoisomerase II-reactive DNA intercalating agents including IDA. This resistance is thought to be secondary to the resistance of the topoisomerase II within HL-60/AMSA cells. Surprisingly, HL-60/AMSA cells were minimally resistant to the cytotoxic and DNA cleaving actions of another anthracycline, doxorubicin (ADR). Comparing other effects of the two anthracyclines revealed that IDA, but not ADR, produced endonucleolytic cleavage, a marker of apoptosis. These results suggest that DNA intercalating anthracyclines can have different effects in human leukemia cells. In the case of IDA, drug actions were similar to those produced by the majority of intercalating agents examined in this cellular system. In the case of ADR, the ability to circumvent the resistance of HL-60/AMSA suggests additional, non-topoisomerase II-mediated mechanisms of cytolysis that may also explain the broad spectrum of clinical activity of ADR.

Activity of two novel anthracene-9,10-diones against human leukemia cells containing intercalator-sensitive or -resistant forms of topoisomerase II

We have examined the activities of two novel aza-anthracene-9,10-diones (aza), 1-aza and 2-aza, in HL-60 human leukemia cell lines containing type II topoisomerases with different sensitivities to inhibition by other intercalating agents. The sensitive line, HL-60, was sensitive to 2-aza but not to 1-aza, whereas the resistant HL-60/AMSA was sensitive to neither agent. Measurements of 1- and 2-aza-induced, topoisomerase II-mediated DNA cross-linking in the cells revealed patterns of resistance and sensitivity that paralleled the results in the cytotoxicity assays. However, measurements of drug-induced topoisomerase II-mediated DNA cross-linking using purified HL-60 and HL-60/AMSA topoisomerase II indicated that both agents could stabilize a covalent complex between DNA and the HL-60 enzyme. HL-60/AMSA topoisomerase II resisted stabilization by either agent. This suggests that the resistance of HL-60 cells to 1-aza is not due to the inability of this drug to inhibit topoisomerase II but rather to another, undefined mechanism.

Effect of transfection of a Drosophila topoisomerase II gene into a human brain tumour cell line intrinsically resistant to etoposide

The human brain tumour cell line HBT20 is intrinsically resistant to etoposide and does not express mdr-1 mRNA. These studies were conducted to determine whether transfecting a Drosophila (D) topoisomerase II (topo II) gene into HBT20 cells could increase their sensitivity to etoposide. A D-topo II construct in a pMAMneo vector under the control of a mouse mammary tumour virus (MMTV) promoter was transfected into HBT20 cells. The gene is inducible by dexamethasone (Dex). The growth rate of the transfected cells and percentage of the cells in G1, S and G2M was no different than the parental cells. Survival after etoposide exposure (10 microM x 2 h) was measured by colony formation. Parental cells and cells transfected by pMAMneo vector alone showed no enhanced etoposide sensitivity after 24 h of Dex stimulation. By contrast, D-topo II transfected cells were sensitised 3-fold when etoposide treatment was preceded by 24 h Dex stimulation. Northern blotting and Western blotting confirmed that Dex had induced D-topo II expression in the sensitised cells. However, in D-topo II-transfected cells increasing the duration of Dex stimulation to 48 h eliminated the sensitisation to etoposide although increased MMTV promoter activity and expression of the D-topo II gene persisted. Measurement of endogenous human topo-II mRNA and protein revealed a decrease after Dex exposure of greater than 24 h. At these distal times, the total cellular topo II levels (endogenous + exogenous) may be decreased, which may explain why increased sensitivity to etoposide could no longer be demonstrated. This model suggests that D-topo II gene transfection can sensitise de novo resistant HBT20 cells to etoposide but that the time frame of that sensitisation is limited.

Further characterization of an amsacrine-resistant line of HL-60 human leukemia cells and its topoisomerase II. Effects of ATP concentration, anion concentration, and the three-dimensional structure of the DNA target.

The characterization of type II topoisomerases from amsacrine-sensitive (HL-60) and amsacrine-resistant (HL-60/AMSA) human leukemia cells was extended. The intercalator resistance and etoposide sensitivity of the HL-60/AMSA cells themselves were confirmed, and the stability of this pharmacologic phenotype over many hundreds of cell generations was demonstrated. Prolonging exposure of HL-60/AMSA cells to amsacrine did not alter their sensitivity relative to that of HL-60 cells. Improved methods of immunoblotting allowed clear demonstration that the topoisomerase II within these cells exhibited sensitivity and resistance characteristics that mirrored those of the cells and the isolated enzymes themselves. Additional biochemical characterization of the type II topoisomerases indicated that both enzymes relaxed supercoiled DNA in a distributive fashion and that the ATP concentrations at which optimal catalytic activity of the two enzymes was exhibited were identical. The enzymes differed, however, in their activity optima in buffers of various type and ionic strength. Furthermore, the inability of the HL-60/AMSA enzyme to exhibit enhanced DNA cleavage in the presence of amsacrine could be overcome if the DNA target molecule contained a bend cloned into its polylinker region. By contrast, a bend in a DNA plasmid containing no polylinker was resistant to amsacrine-enhanced cleavage in the presence of HL-60/AMSA topoisomerase II, as was a plasmid containing a polylinker with no bend. This suggests that an unusual DNA conformation (a bend) in a specific DNA context (a polylinker) may be a favored site for topoisomerase II action. It also suggests a mechanism by which the sites and extent of topoisomerase II activity can be controlled in cells.

A restriction fragment length polymorphism for human topoisomerase II: Possible relationship to drug-resistance

In previous studies we used Southern blotting to examine the topoisomerase II locus (on chromosome 17) in human leukemia cell lines and noted a difference in the XmnI restriction endonuclease digestion pattern between an m-AMSA-resistant line and its m-AMSA-sensitive parent line (Zwelling, L. A.; Hinds, M,; Chan, D.; Mayes, J.; Sie, K. L.; Parker, E.; Silberman, L.; Radcliffe, A.; Beran, M.; Blick, M. Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II. Journal of Biological Chemistry 264:16411-16420; 1989). We now demonstrate that the variable XmnI digestion pattern represents a normal restriction fragment length polymorphism (RFLP) which is observed in subjects without malignant disease and exhibits an autosomal pattern of inheritance. These data suggest that the previously described deviation in the genomic structure of topoisomerase II in the m-AMSA-resistant cell line did not reflect a new mutation, but rather a reduction to homozygosity at the topoisomerase II locus. This reduction to homozygosity is not due to chromosomal loss, as chromosome 17-specific gene probes clearly identify two chromosome 17s in the sensitive line and four in the resistant line, using chromosome painting with a chromosome 17-specific library. Some other genetic change must be the cause of the resistance of HL-60/AMSA and its topoisomerase II to the inhibiting actions of m-AMSA.

Anion-dependent modulations of DNA topoisomerase II-mediated reactions in potassium-containing solutions

DNA binding proteins operate in an intracellular environment of low chloride concentration, yet in vitro assays of the activities of these proteins are often performed in isotonic chloride-containing solutions. Previously, the activity of bacterial DNA-binding proteins was found to be enhanced in potassium-containing solutions in which the anion glutamate (Glu) was substituted for chloride. We have extended this observation to include eukaryotic topoisomerase I and II activities. The concentration ranges over which DNA strand passing activities of these enzymes were observed was broader in KGlu than in KCl. This was also true for the topoisomerase II-mediated DNA strand passage and antineoplastic drug-dependent DNA cleavage produced by nuclear extracts from HL-60 human leukemia cells. The rate of topoisomerase II-mediated DNA strand passage was also dependent on anion moiety and concentration in potassium-containing buffers. Drug-dependent topoisomerase II-mediated DNA cleavage in intact HL-60 cell nuclei was also anion-dependent, suggesting that anion type and concentration may influence topoisomerase II-mediated events in mammalian cells as had been described for other DNA binding proteins in prokaryotic systems. This should be considered in developing biochemical assays of topoisomerase activities to reproduce intracellular conditions.

Phorbol regulation of topoisomerases I and II in human leukemia cells. Studies in an additional cell pair sensitive or resistant to phorbol-induced differentiation

We previously reported (Zwelling et al., Cancer Res 50: 7116-7122, 1990) that etoposide-induced DNA cleavage and mRNA coding for topoisomerase II are reduced in HL-60 cells induced to differentiate by phorbol ester. Reduction of etoposide-induced cleavage and topoisomerase II message did not occur in the derived cell line 1E3 (which is resistant to phorbol-induced differentiation), implying that topoisomerase II activity may be related to the state of cell differentiation. We have extended these studies using a new phorbol sensitive/resistant cell pair, S (sensitive) and PET (phorbol ester tolerant). Phorbol ester exposure not only reduced etoposide-induced DNA cleavage and topoisomerase II mRNA in S cells but also decreased the amount of immunoreactive topoisomerase II enzyme in whole S cells. However, immunoreactive topoisomerase II extracted from the nuclei of phorbol-treated S cells was not reduced compared with that from the nuclei of untreated S cells. This suggests that topoisomerase II contained in nuclear extracts is not always representative of the total cellular enzyme. Dramatic decreases in the amount, activity, or gene expression of topoisomerase II were not observed after phorbol treatment of the resistant PET cells; this is consistent with the potential involvement of topoisomerase II in monocytoid differentiation. Levels of topoisomerase I enzyme and mRNA fell in both S and PET cells after phorbol treatment; therefore, the genes for topoisomerases I and II did not appear to be regulated coordinately.

The effect of staurosporine on drug-induced, topoisomerase II-mediated DNA cleavage in human leukemia cells

SummaryPhorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C, dramatically decreased topoisomerase II-reactive drug-induced DNA cleavage in HL-60 human cells. The effect of staurosporine, an inhibitor of protein kinase C, on drug-induced, topoisomerase II-mediated DNA cleavage was quantified in the same cells. Staurosporine decreased the magnitude of 4′-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)- and etoposide-induced DNA cleavage in a dose-and time-dependent fashion. Measurement of several parameters of cell proliferation revealed no clear and uniform correlation between staurosporines inhibition of these parameters and its effects on drug-induced DNA cleavage. A direct comparison with PMAs effects on drug-induced DNA cleavage showed that whereas PMAs inhibition of etoposide-induced cleavage was much greater than its inhibition ofm-AMSA-induced cleavage, the magnitude of staurosporines effect on the cleavage produced by the two topoisomerase II-reactive drugs was similar. Thus, although PMA stimulates protein kinase C and staurosporine inhibits this enzyme, it is unlikely that the actions of either on topoisomerase II-reactive, drug-induced DNA cleavage are mediated directly via protein kinase C. Furthermore, it is likely that the mechanisms by which PMA and staurosporine inhibit topoisomerase II-reactive drug-induced cleavage are different.

Effect of bryostatin 1 on drug-induced, topoisomerase II-mediated DNA cleavage and topoisomerase 11 gene expression in human leukemia cells

Unlike PMA, bryostatin 1 has been found to have a minimal effect on drug-induced topoisomerase II-mediated DNA cleavage and no effect on topoisomerase II mRNA levels. Furthermore, bryostatin 1 overcame the down-regulatory effects of PMA treatment on (1) drug-induced, topoisomerase II-mediated DNA cleavage, (2) drug-induced cytotoxicity, and (3) topoisomerase II gene expression. Thus, it is unlikely that the effects of phorbol ester treatment on topoisomerase II-mediated events are a direct consequence of protein kinase C activation per se. Rather, the results with bryostatin 1 suggest that the phorbol ester effects are related to more distal effects of phorbol ester treatment that may be related to monocytoid differentiation.