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.     

Modulation of DNA topoisomerase II activity and expression in melanoma cells with acquired drug resistance

The role of DNA topoisomerases (Topo) IIalpha and IIbeta was investigated in various drug-resistant melanoma cells. Melanoma cells resistant to etoposide, exhibited an up to tenfold reduced Topo II activity corresponding to an increasing degree of drug resistance indicating that modulation of Topo II activity contribute to the drug-resistant phenotype. The reduction of Topo II activity was reflected by decreased nuclear amounts of both Topo II isoforms.     

Mechanisms of resistance to DNA topoisomerase II inhibitors]

Cytotoxicity of anticancer drugs are depended on cellular differences of drug transport processes, drug metabolism or proliferation process including DNA synthesis. Thus, resistance to anticancer drugs is likely due to many cellular changes. Mechanisms of resistance to anticancer drugs have been studied with cells selected for their ability to grow in the presence of drugs. In this paper, the mechanisms of cellular resistance to DNA topoisomerase II inhibitors was described.     

Expression and integrity of DNA topoisomerase II isoforms does not explain generic drug resistance in malignant mesothelioma

PURPOSE: Malignant mesothelioma is a tumour that is highly resistant to a number of different chemotherapy agents, yet the mechanisms by which resistance occurs are poorly understood. The pattern of resistance is consistent with disruption of topoisomerase function or expression. Coupled with this, we have previously noted a common serological reaction to the beta isoform of topoisomerase II, suggesting that it may be aberrantly expressed in patients with mesothelioma. METHODS: We assessed the expression of topoisomerase II isoforms in sections of primary tumour. We tested a panel of five mesothelioma cell lines for sensitivity to the known topoisomerase-targeting drugs, doxorubicin and etoposide. We sequenced expressed segments of the topoisomerase genes from these cell lines that have previously been associated with drug resistance. We then investigated other potential resistance mechanisms. RESULTS: We found that the beta isoform of topoisomerase II was more frequently expressed in primary tumours. Only one of the five cell lines was highly resistant to etoposide and this cell line was found to have a point mutation in the gene for topoisomerase IIalpha. Protein levels of topoisomerase IIalpha and beta did not correlate with sensitivity to either doxorubicin nor to etoposide. Semiquantitative analysis suggested that there was marked variation in the levels of mRNA expression of MRP, gamma-GCS and MDR1. None of these findings could be associated with resistance to chemotherapy. CONCLUSION: We conclude that mutations in topoisomerase IIalpha can be associated with extreme resistance of mesothelioma to etoposide. The generic drug resistance of this tumour requires further investigation.     

Enhanced expression of DNA topoisomerase II by recombinant human granulocyte colony-stimulating factor in human leukemia cells.

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on DNA topoisomerase II (topo II) expression was studied in two human acute myelogenous leukemia cell lines, NKM-1 and NOMO-1, which express G-CSF receptor and proliferate in response to exogenous G-CSF. Northern blot analysis revealed that the level of topo II mRNA in 16-h stimulated cells in serum-free medium with G-CSF (10 ng/ml) was approximately 2-fold higher than that in cells without G-CSF. Enhanced topo II mRNA expression was detectable within 3 h after the addition of G-CSF. Topo II activity in crude nuclear extracts from 16-h G-CSF-stimulated cells was also found to be approximately 2-fold greater than that from unstimulated cells. According to in vitro cytotoxic assay, the sensitivity of G-CSF-stimulated cells to intercalating (daunorubicin) and nonintercalating (etoposide) topo II-targeting drugs increased significantly, whereas no enhancement of sensitivity was observed with an alkylating agent (4-hydroperoxycyclophosphamide). The augmented drug sensitivity observed was not due to the increased level of drug transport, as suggested by the similar extent of [3H]etoposide uptake between G-CSF-stimulated and unstimulated cells. By measuring the topo II mRNA and the cytotoxicity of the above mentioned drugs, we obtained essentially the same results in G-CSF-responsive leukemia cells isolated from three acute myeloblastic leukemia patients, as observed in the cultured cell lines. These findings strongly suggest that the sensitivity to "topo II-targeting drugs" could be augmented by exogenous G-CSF through elevated topo II activity in G-CSF-responsive leukemia cells.     

Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells

The intracellular level of DNA topoisomerase II appears to be reversibly regulated by serum concentration in cultured primary human skin fibroblasts (HSF). Upon serum starvation, the intracellular level of topoisomerase II in HSF, as monitored by immunoblotting with antitopoisomerase II antibodies, gradually decreased to a nondetectable level (less than 10(4) copies/cell) over a period of 72 h. Addition of 10% serum to the starved cells led to a gradual increase of the intracellular topoisomerase II to the original level (approximately 10(6) copies/cell) over a period of 24 h. The intracellular DNA topoisomerase II level in HSF is also sensitive to cell density; minimally a 7-fold decrease was observed when HSF were grown to saturation density in a constant serum concentration. Similarly, the intracellular levels of DNA topoisomerase II in other "nontransformed" cells such as mouse NIH 3T3 and 3T6 cells are also sensitive to both the serum concentration and the cell density. In contrast, topoisomerase II levels in transformed cells such as HeLa cells, L1210 cells, and SV40 T-antigen-transformed COS-1 cells are maintained at high levels (approximately 10(6) copies/cell) and are much less sensitive to growth conditions. The topoisomerase II level in HeLa cells synchronized by a double thymidine block remained relatively constant (less than 2-fold difference) throughout the late G1, S, G2, and M phases of the cell cycle. Our results suggest that the level of DNA topoisomerase II is primarily regulated in the G0-G1 phase of the cell cycle and is elevated to a high level (approximately 10(6) copies/cell) in proliferating cells. In contrast, the intracellular levels of DNA topoisomerase I in these cells were largely unaffected by these growth conditions either in HSF or in HeLa cells.     

Resveratrol acts as a topoisomerase II poison in human glioma cells

Recently, we demonstrated that Resveratrol (RSV), a well known natural stilbene, is able to induce a delay in S progression with a concomitant increase in γH2AX expression in U87 glioma cells. Furthermore, we showed that it inhibits the ability of recombinant human topoisomerase IIα to decatenate kDNA in vitro. Because proliferating tumor cells express topoisomerases at high levels and these enzymes are important targets of some of the most successful anticancer drugs, we tested whether RSV is able to poison topoisomerase IIα in glioma cells. Then, we monitored the increase of micronuclei in RSV treated U87 cells as a consequence of the conversion of TOPOII/DNA cleavable complexes to permanent DNA damage. Finally, we assayed the ability of RSV in modulating the expression of target proteins involved in DNA damage signalling, namely ATR, ATM, Chk1, Chk2 and γH2AX. Through a molecular modelling here we show that RSV binds at the TOPOII/DNA interface thus establishing several hydrogen bonds. Moreover, we show that RSV poisons TOPOIIα so inducing DNA damage; ATM, Chk2 and γH2AX are involved in the DNA damage signalling after RSV treatment.     

Reduction of drug accumulation and DNA topoisomerase II activity in acquired teniposide-resistant human cancer KB cell lines

We have isolated stable teniposide (VM26)-resistant cell lines from human cancer KB cells by stepwise exposure to increasing doses of the drug. At each step, we have purified VM26-resistant cell lines. KB/VM-a, KB/VM-b, KB/VM-1, KB/VM-2, KB/VM-3, and KB/VM-4 showed 3-, 6-, 12-, 16-, 74-, and 95-fold higher resistance to VM26 than did KB. We have further characterized KB/VM-2 and KB/VM-4 which showed about 15- and 100-fold higher resistance to VM26 or etoposide (VP16) than did KB. Both VM26-resistant cell lines showed 4- to 11-fold higher relative resistance to daunomycin and Adriamycin than did KB. Steady-state levels of the cellular accumulation of radioactive VP16 in KB/VM-2 and KB/VM-4 cells were about 40% of that of KB cells, whereas similar levels of radioactive daunomycin accumulation were observed in KB/VM-2 and KB/VM-4 cells as KB cells. Topoisomerase II activity of nuclear extracts of both KB/VM-2 and KB/VM-4 assayed by decatenation of kinetoplast DNA was consistently two-thirds or less the activity of KB cells. A similar reduction was seen in both immunoblot assays with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA. DNA topoisomerase I activity, however, was similar between the mutants and their parent. Furthermore, cell growth of KB/VM-2 and KB/VM-4 was more thermolabile than that of KB, while KB/VM-b already showed temperature-sensitive growth. KB/VM-1 did show reduced accumulation of VP16 as in KB/VM-2 or KB/VM-4, but it had a normal level of topoisomerase II content as in KB cells. These data suggest that the reduced expression of DNA topoisomerase II, possibly combined with decreased permeability to the drugs, can account for the acquired VM26 resistance of KB/VM-2 and KB/VM-4 cells and also that the temperature-sensitive phenotype might not be obligatorily coupled with the reduced expression of topoisomerase II or the decreased permeability.     

Epidermal growth factor induction of resistance to topoisomerase II toxins in human squamous carcinoma A431 cells

Alteration of the epidermal growth factor (EGF) signaling pathway occurs frequently in human cancer cells and may subsequently affect the cell survival towards anti-cancer agents. To elucidate the effect of long-term EGF treatment on the chemo-sensitivity of human cancer cells, human squamous carcinoma A431 cells (AP) were incubated continuously with 50 ng/ml EGF for 30 weeks and these cells were designated as the AC cells. The long-term EGF treatment did not alter the EGFR level and the EGF-induced protein tyrosine phosphorylation pattern in the AC cells. By MTT assay, the AC cells were shown to be more resistant than the AP cells to doxorubicin, etoposide and amsacrine but not to cisplatin. Among the drug-resistant proteins, topoisomerase IIalpha (topoII) was downregulated in the AC cells while there was no apparent change in the levels of P-glycoprotein, MRP-1 or glutathione- S-transferase-pi as compared to the AP cells. Furthermore, knockdown of topoII by antisense topoII oligonucleotide transfection decreased the sensitivity to doxorubicin, etoposide and amsacrine in the A431 cells. Results from the present study support an idea that long-term treatment with EGF may induce drug resistance in cells through the downregulation of topoII.     

DNA topoisomerase I and II expression in drug resistant germ cell tumours

A small number of testicular germ cell tumours are refractory to current chemotherapy regimens. DNA topoisomerase I is the target for several new drugs and a potential candidate treatment for chemorefractory germ cell tumours. DNA topoisomerase II alpha is the target for etoposide, which is currently used regularly in germ cell tumour treatment. The expression of DNA topoisomerase I and II alpha were therefore assessed immunohistochemically in a range of testicular tumours, especially those with persistent malignant elements on retroperitoneal lymph node dissection. Pre-chemotherapy orchidectomy specimens were matched with post-chemotherapy retroperitoneal lymph node dissections to examine changes in expression. There was considerable variation in the expression of topoisomerase I in different tumour types. Both yolk sac tumours and teratoma, mature showed universal expression of topoisomerase I, while 38% of seminomas and 30% of embryonal carcinomas were positive. Strong topoisomerase II alpha expression was found in embryonal carcinoma. There was a negative correlation between topoisomerase I and II alpha expression (P=0.004) and downregulation of topoisomerase II alpha after chemotherapy (P=0.02). Topoisomerase I expression appears to increase in those cases with residual teratoma, mature, but is largely unchanged in those cases remaining as embryonal carcinoma. These results suggest that topoisomerase I inhibitors may be useful in chemorefractory germ cell tumours, especially yolk sac tumours and where there are unresectable residual teratoma, mature deposits.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide.

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell''s sensitivity to etoposide.     

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIα catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIα to decatenate kDNA, so that it could be considered a TOPO II poison.     

DNA topoisomerase II immunostaining in human leukemia and rhabdomyosarcoma cell lines and their responses to topoisomerase II inhibitors.

DNA topoisomerase II is an enzyme that affects nuclear structure and function and is the target of a number of anticancer drugs in clinical use, including teniposide (VM-26). We have used our polyclonal antisera that recognize both the M(r) 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II in cytospin preparations of drug-sensitive (CEM) and VM-26-resistant (CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. We have also examined the nuclear distribution of topoisomerase II in monolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. In the absence of drug, we observed a focal "patchy" staining of nuclear topoisomerase II in all cell lines, that was especially notable in the lymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26 under conditions that increase the number of covalent topoisomerase II-DNA complexes increased both the intensity and the homogeneity of nuclear topoisomerase II staining in a subpopulation of cells; focal staining was less evident after treatment with drug. These responses were roughly proportional to the concentration of VM-26 used and required only brief (approximately 25-min) incubation with drug. We also found that treatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-anisidide similarly increased the intensity and homogeneity of nuclear topoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)methanesulfon-o-anisidide and 1-beta-D-arabinofuranosylcytosine, agents that do not inhibit topoisomerase II, did not produce this effect. Finally, the VM-26-mediated alteration in topoisomerase II staining intensity and distribution was attenuated in proportion to the degree of VM-26 resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNA-topoisomerase covalent ("cleavable") complexes in intact cells. Our findings indicate that anti-topoisomerase II drugs, such as VM-26, have profound effects on the ability to detect topoisomerase II in the nucleus and provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.     

Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines

In a previous study, we established camptothecin (CPT)-resistant cell lines, A549/CPT and HT-29/CPT, from human lung cancer A549 and human colon cancer HT-29. A549/CPT was shown to express similar amounts of DNA topoisomerase I (topo I) as the parental line, and HT-29/CPT was shown to express lower amounts of topo I than its parental line. DNA topoisomerases I and II are known to be functionally related. In the present study, the possible alterations in topo II expression were examined in these human CPT-resistant lines. In A549/CPT and HT-29/CPT, the cellular contents of topo II and its mRNA were elevated over that seen in each parental line. Nuclear extracts from A549/CPT and HT-29/CPT showed higher topo II activity than those from the corresponding parental lines when the same amounts of nuclear protein were used. Topo II was partially purified from HT-29 and HT-29/CPT by hydroxylapatite column chromatography, and the enzyme activities were compared. HT-29/CPT showed higher topo II activity in the hydroxylapatite column-eluted fractions than HT-29. These results indicate the possible activation of topo II expression in the CPT-resistant cell lines.     

RAD51 as a potential surrogate marker for DNA repair capacity in solid malignancies

Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors, and increased DNA repair capacity is a hallmark mechanism of resistance not only to DNA‐damaging treatments such as cytotoxic drugs and radiotherapy, but also to small molecule targeted drugs such as inhibitors of poly‐ADP ribose polymerase (PARP). Hence, there is substantial medical need for potent and convenient biomarkers of individual response to DNA‐targeted treatment in personalized cancer care. RAD51 is a highly conserved protein that catalyzes DNA repair via homologous recombination, a major DNA repair pathway which directly modulates cellular sensitivity to DNA‐damaging treatments. The clinical and biological significance of RAD51 protein expression is still under investigation. Pre‐clinical studies consistently show the important role of nuclear RAD51 immunoreactivity in chemo‐ and radioresistance. Validating data from clinical trials however is limited at present, and some clinical studies show controversial results. This review gives a comprehensive overview on the current knowledge about the prognostic and predictive value of RAD51 protein expression and genetic variability in patients with solid malignancies.     

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.     

Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors

Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9-acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide.