Expression Of Topoisomerase Research Articles

BIRICODAR (VX-710; Incel™): an effective chemosensitizer in neuroblastoma

Clinical studies have suggested that both MDR1 and MRP may play a significant role in the chemosensitivity and outcome of neuroblastoma. To clarify the nature of multidrug resistance (MDR) in this tumour a series of six neuroblastoma cell lines have been characterized with regard to P-glycoprotein, MRP and LRP expression using immunocytochemistry and expression of MDR1, MRP, LRP and topoisomerase II genes using reverse transcription polymerase chain reaction (RT-PCR). By RT-PCR, all lines expressed MRP, five expressed LRP and four expressed MDR1, but protein levels of each of these were variable. Chemosensitization to a range of MDR-associated drugs (vincristine, doxorubicin, etoposide, taxotere, topotecan) and non-MDR-associated drugs (cisplatin, melphalan) by three modulating agents, cyclosporin A, PSC 833 and the novel Biricodar (VX-710; Incel™), was evaluated using a colourimetric cytotoxicity assay (MTS). Alteration of daunorubicin efflux by these agents was evaluated using FACS analysis. Clonogenic assay was used to study the influence of these chemosensitizers on vincristine cytotoxicity. Marked sensitization to vincristine was observed in MDR1-positive lines, and a similar but less consistent effect was seen with taxotere, doxorubicin and etoposide. With MRP-positive, MDR-negative lines, only VX-710 caused consistent sensitization. These data confirm MDR1 and MRP expression as contributory factors in chemoresistance in neuroblastoma and indicate that VX-710 may be a useful modulator of both mechanisms and worthy of clinical evaluation in this tumour.

BIRICODAR (VX-710; Incel): an effective chemosensitizer in neuroblastoma.

Clinical studies have suggested that both MDR1 and MRP may play a significant role in the chemosensitivity and outcome of neuroblastoma. To clarify the nature of multidrug resistance (MDR) in this tumour a series of six neuroblastoma cell lines have been characterized with regard to P-glycoprotein, MRP and LRP expression using immunocytochemistry and expression of MDR1, MRP, LRP and topoisomerase II genes using reverse transcription polymerase chain reaction (RT-PCR). By RT-PCR, all lines expressed MRP, five expressed LRP and four expressed MDR1, but protein levels of each of these were variable. Chemosensitization to a range of MDR-associated drugs (vincristine, doxorubicin, etoposide, taxotere, topotecan) and non-MDR-associated drugs (cisplatin, melphalan) by three modulating agents, cyclosporin A, PSC 833 and the novel Biricodar (VX-710; Incel™), was evaluated using a colourimetric cytotoxicity assay (MTS). Alteration of daunorubicin efflux by these agents was evaluated using FACS analysis. Clonogenic assay was used to study the influence of these chemosensitizers on vincristine cytotoxicity. Marked sensitization to vincristine was observed in MDR1-positive lines, and a similar but less consistent effect was seen with taxotere, doxorubicin and etoposide. With MRP-positive, MDR-negative lines, only VX-710 caused consistent sensitization. These data confirm MDR1 and MRP expression as contributory factors in chemoresistance in neuroblastoma and indicate that VX-710 may be a useful modulator of both mechanisms and worthy of clinical evaluation in this tumour. © 1999 Cancer Research Campaign

The Topoisomerase-related Function Gene TRF4 Affects Cellular Sensitivity to the Antitumor Agent Camptothecin

Camptothecin is an antitumor agent that kills cells by converting DNA topoisomerase I into a DNA-damaging poison. Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood. We report here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair camptothecin-mediated damage to DNA. The hypersensitivity to camptothecin in the trf4 mutant does not result from elevated expression of DNA topoisomerase I. We show that Trf4 is a nuclear protein whose expression is cell cycle-regulated at a post-transcriptional level. Suppression of camptothecin hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may influence higher order chromosome structure, ZDS1 and ZDS2. We have isolated and sequenced two human TRF4 family members, hTRF4-1 and hTRF4-2. The hTRF4-1 gene maps to chromosome 5p15, a region of frequent copy number alteration in several tumor types. The evolutionary conservation of TRF4 suggests that it may also influence mammalian cell sensitivity to camptothecin.

V‐src induces cisplatin resistance by increasing the repair of cisplatin–DNA interstrand cross‐links in human gallbladder adenocarcinoma cells

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3–1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3–1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway. Int. J. Cancer 80:731–737, 1999. © 1999 Wiley-Liss, Inc.

V-src induces cisplatin resistance by increasing the repair of cisplatin-DNA interstrand cross-links in human gallbladder adenocarcinoma cells.

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

Plasmodium falciparum:Stage-Related Expression of Topoisomerase I

Tosh, K., Cheesman, S., Horrocks, P., and Kilbey, B. 1999.Plasmodium falciparum:Stage-related expression of topoisomerase I.Experimental Parasitology91,126–132. The expression and activity of topoisomerase I (PfTopoI) has been examined during the intraerythrocytic stages of thePlasmodium falciparumlife cycle. The promoter is inactive during the early ring stage and becomes active only during the later trophozoite and schizont stages. The PfTOP1transcript starts to accumulate in the trophozoite stage parasite, decreasing again in the schizont stage. Using both stage-specific Western analysis and immunofluorescent assays we show that PfTopoI is present at low levels in rings and accumulates to approximately equal levels in the trophozoite and schizont stages. Experiments to determine the activity of PfTopoI, using a topoisomerase I relaxation assay, show that there is a low level of PfTopoI activity in both ring and trophozoite stages, but activity increases dramatically in the schizont stage. The PfTopoI activity can be inhibited by treatment with specific antiserum and by the type I topoisomerase-specific inhibitor camptothecin.

胎児期における小脳皮質の発生とDNAトポイソメラーゼIおよびIIβの発現

胎児期における小脳皮質の発生とDNAトポイソメラーゼIおよびIIβの発現

Increased phosphorylation of DNA topoisomerase II in etoposide resistant mutants of human glioma cell line.

The efficacy of the epipodophyllotoxins VP-16 and VM-26 is limited by the occurrence of drug resistance in the tumor cell population. Cellular insensitivity to drugs that stabilize the cleavable complex is frequently expressed as multidrug resistance (MDR). In some cell lines, overexpression of MDR-1/P-glycoprotein or the multidrug resistance associated protein (MRP) has been demonstrated and implicated as the mechanism of resistance. Typically, these cells have reduced drug accumulation, secondary to increased drug efflux. In other cell lines, an atypical MDR phenotype has been identified, with the predominant mechanism of resistance shown to be qualitative and/or quantitative changes in the levels and activity of topoisomerase II. For VP-16, increased expression of MDR-1 or MRP and alterations in topoisomerase II have been shown to confer tolerance. To further understand resistance to VP-16, T98G-VP(1000) was initially isolated as a single clone from parental cell, T98G, by exposure to VP-16. Subsequently, a population of cells from this subline was exposed to three-fold higher drug concentration allowing stable sublines to be established at higher extracellular drug concentration. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolate. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 47-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased, with increased VP-16 efflux and reduced accumulation. This adaptation allowed for partial restoration of topoisomerase II activity secondary to increased expression and hyperphosphorylation, with a resultant increase in growth rate. In this cell line, hyperphosphorylation coincided with increased casein kinase II mRNA protein levels, without increased PKC protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase II protein levels secondary to an acquired 615 bp deletion in one topoisomerase II allele, which resulted in reduced protein levels. In this subline, high levels of resistance were attained as a result of synergism between the reduced topoisomerase II levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from additional mechanisms helping to confer high levels of resistance.

Differential expression of DNA topoisomerase II and isozymes in human ovarian cancer

Chien C-H, Chow S-N, Yang C-H, Ng H-T, Lin C-T. Differential expression of DNA topoisomerase II α and β isozymes in human ovarian cancer. Int J Gynecol Cancer 1998; 8:451–459. The relationship between DNA topoisomerase II (Topo II) expression and human ovarian cancer has been evaluated by analyzing the enzyme activity, enzyme levels, and expression of mRNA of DNA Topo II isozymes, α and β, in normal (n = 4), benign (5 serous cystadenomas), and malignant (29 serous adenocarcinomas, stages I–III) human ovarian cells, selected from G2M-S phase fractions of similar numbers of cells in the corresponding tissues. By the P4 DNA-unknotting assay, the elevation of DNA Topo II α activity in 29 cases was found to be 2 to 3 times greater than that of normal ovarian tissues. An average 2- to 4-fold increase in the expression of the immunoreactive DNA Topo II α was also revealed by immunoblot analysis in G2M-S phase fractions of cells in the 29 cases of ovarian cancer. A good correlation between catalytic activity of the enzyme and its level in the corresponding tumor tissue was also found. Topo II mRNA levels were found by northern blot coupled with RT-PCR analysis to be increased significantly. An average 5.2–7.2 fold increase of DNA Topo II α mRNA was found in the corresponding cases of stage I–III G2M-S phase fractions of ovarian adenocarcinomas, compared to normal matched ovarian tissue. Eight of 29 malignant ovarian cancer tissues did not show significantly elevated expression of α isoform. No significant changes were found in the activity and expression of β isozyme in various stages of ovarian cancer. The results suggest that the increase in enzyme levels in some advanced ovarian cancer may result from either increased transcription of Topo II α gene or increased mRNA stability. It also suggests the potential usefulness of chemotherapeutic agents targeting DNA Topo II α expressed in some ovarian cancers.

Expression of DNA topoisomerase I, DNA topoisomerase II-alpha, and p53 in metastatic malignant melanoma

New anticancer drugs that target DNA topoisomerase I (topo I) are showing activity against a wide variety of solid human neoplasms. These drugs work by a novel mechanism of action and cause topo I-mediated DNA breaks. These DNA breaks become lethal in cycling cells when they interact with the replication fork. Because of the challenges in treating metastatic malignant melanoma, we performed an immunohistochemical study of this group of neoplasms to search for the presence of molecular markers that might indicate tumor response to topo I active drugs. Using a new immunohistochemical stain for topo I, we found elevation of this protein in 10 of 24 cases (41.6%) of metastatic malignant melanoma. The metastatic tumors that showed increased expression of topo I (2+ or 3+) had statistically significant higher proliferation indices, measured by immunohistochemical staining for DNA topo II-alpha, than did metastatic lesions with no detectable topo I expression. The average topo II-alpha index of metastatic melanomas with 2+ topo I expression was 45.1 (SD = 17.9) and with 3+ topo I expression was 52.3 (SD = 32.5). These values were found to be statistically different (P = .05) than the average topo II-alpha index of 18.9 (SD = 17.7) found for metastatic melanomas without detectable topo I immunostaining. Immunohistochemical staining for p53 suggested abnormal p53 function in 6 of the 10 melanomas (60%), which showed elevations of topo I (2 to 3+ topo I immunostaining) but normal p53 function in all 14 metastatic lesions that showed normal topo I expression.

Altered Expression and Activity of Topoisomerases During All-Trans Retinoic Acid-Induced Differentiation of HL-60 Cells

Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Altered Expression and Activity of Topoisomerases During All-Trans Retinoic Acid-Induced Differentiation of HL-60 Cells

Regulation of topoisomerase II (TOPO II) isozymes α and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIα and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (α and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (α + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Molecular biological evidences for the genetic stability of doxorubicin resistant cell line S-180Rin vivo

Objective: In order to assess the genetic stability of doxorubicin resistance sarcoma S-180R cell linein vivo. Methods: The drug resistant genes and molecules were examined by flow cytometry, Southern blot, Northern blot and RT-PCR. Results: The results showed that drug-efflux in S-180R increased nearly 100-folds, as compared with its parent cells, the rate of half peak width resistant cell/peak high decreased from 0.56 to 0.23 measured by flow cytometry after two years. The mdr1 gene amplified and overexpressed significantly in S-180R and the expression of topoisomerase II α gene decreased remarkably in S-180R. There was no significant different of the MRP expression between S-180R and S-180. Conclusion: These results indicated that drug resistance of S-180R was maintained and also increased. The major mechanism of drug resistance is the amplification and overexpression of mdr1 gene, the decreased expression of topoisomerase II α also contributed to it. So, S-180R is an ideal experimental model for the study of doxorubicin resistance and its reversionin vivo.

Nodular lesions of monocytic component in myelodysplastic syndrome.

We report 4 unusual cases of myelodysplastic syndrome with distinct persistent nodular lesions noted on serial bone marrow examinations, even during remission. The lesions were predominantly composed of immature monocytes that stained positively for CD68. Trisomy 9 and 11 were demonstrated in the cells of the nodular lesions and surrounding marrow of 1 patient, indicating the same clonal origin. Evaluation of p53 glycoprotein, retinoblastoma protein (pRb), proliferation-related protein (Ki-67), multiple drug-resistant enzyme glutathione-S-transferase pi, and topoisomerase IIalpha (Topo IIalpha) revealed decreased topoisomerase expression within the nodular lesions compared with the surrounding marrow and absence of Ki-67 antigen within nodular lesions. Most cells in the lesion were not in a proliferative cycle, with very low expression of Topo IIalpha, which may explain the apparent drug resistance of these nodular lesions.

Induction of telomerase activity in stimulated human lymphocytes precedes expression of topoisomerase II α

Telomerase activity has been demonstrated in human immortal cell lines and in tumors, whereas it is generally absent from normal tissues, with the exception of germ cells. Low levels have also been detected in blood and skin cells. In this report we describe up-regulation of telomerase activity in normal human blood lymphocytes by mitogen stimulation. After 24 h of mitogen treatment a strong induction was detectable using the PCR-based telomeric repeat amplification protocol. The level of activity remained almost constant when the cultivation lasted 72 h. By contrast, topoisomerase II alpha was induced later with a maximum expression after 48-72 h. Our data show that telomerase can be induced in normal peripheral lymphocytes prior to expression of the S-phase typical protein topoisomerase II alpha indicating that telomere elongation might be initiated before DNA replication.

Intraerythrocytic expression of topoisomerase II from Plasmodium falciparum is developmentally regulated

The stage-specific relationship between promoter activity, transcript production, protein expression and enzyme activity has been investigated for the gene encoding Plasmodium falciparum topoisomerase II (PfTopoII). Nuclear run-on experiments have shown that the P. falciparum topoisomerase II gene (Pf TOP2) promoter is active at low levels in ring stage parasites, but reaches high levels of activity as the parasites progress into trophozoite/schizont asexual stages. Steady-state Pf TOP2 transcripts are present at low levels in rings, accumulate in trophozoites, but are completely undetectable in schizonts. An antiserum raised against the species-divergent carboxy-terminus of PfTopoII, which neutralised the decatenation activity in parasite extracts, was used to probe Western blots of ring, trophozoite and schizont stage parasite extracts. Relatively low levels of PfTopoII were seen in rings compared with those in trophozoite and schizont preparations. Parasite extracts were also used to compare the patterns of protein accumulation and enzyme activity at these stages. Complete decatenation of kinetoplast substrate DNA (KDNA) was found in schizont stages, very low levels of activity were observed in rings and trophozoites showed intermediate levels. These finding show that, as parasites progress towards the stages where DNA replication occurs, there is a concomitant increase in both topoisomerase II production and activity.

The expression of topoisomerase II α and Ki-67 in the gastric carcinoma

The expression of topoisomerase II α and Ki-67 in the gastric carcinoma

Drug resistance to topoisomerase II inhibitors

We describe in this review the mechanisms of resistance to topoisomerase II inhibitors that have been identified in cell lines rendered resistant to drugs. They concern especially both quantitative and qualitative alterations of topoisomerase II, leading to drug insensitivity of the cells. Expression and activity of topoisomerase II have also been studied in a number of tumor specimens originating from patients, but the role of topoisomerase II alterations in drug resistance in the clinical setting has not yet been firmly established. It would be worthwhile, however, to develop predictive assays for drug activity in human cancers, based upon the topoisomerase II status of tumor samples.

The bis(naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic topoisomerase II.

DMP 840 ((R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2, 1-ethanediyl)]-bis(5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione] dimethanesulfonate) is a novel bis(naphthalimide) that has shown promising antitumor activity in a variety of preclinical model systems. The compound binds to DNA with high affinity and intercalates, but the mechanism of cell killing has not been elucidated. We have used yeast strains to test whether DMP-840 is active against either topoisomerase I or II. We found that temperature-sensitive top2 mutants resistant to etoposide or amsacrine also confer resistance to DMP-840. In addition, cells overexpressing yeast topoisomerase II were hypersensitive to the drug. By contrast, top1 deletions rendered cells hypersensitive to the drug. These results strongly suggest that DMP-840 acts against eukaryotic topoisomerase II and kills cells by converting the enzyme into a cellular poison. We verified that DMP-840 is active against eukaryotic topoisomerase II by demonstrating that the drug stimulates formation of a cleavage complex with purified yeast topoisomerase II in vitro. We also demonstrated that the drug is active against human topoisomerase II by showing that expression of human topoisomerase II restored sensitivity of resistant yeast cells to DMP-840. We have also directly demonstrated that DMP-840 acts as a poison against purified human topoisomerase II alpha. Taken together, these results indicate that DMP-840 acts like other intercalating topoisomerase II poisons; it kills eukaryotic cells by stabilizing the cleavage complex of topoisomerase II with DNA.

Expression of DNA topoisomerases in cerebellar development of rat embryos

Expression of DNA topoisomerases in cerebellar development of rat embryos