An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene.

We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 µM) and 72.9% (8 µM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 µM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria.

A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.

Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity.

Anticancer effect of SZC017, a novel derivative of oleanolic acid, on human gastric cancer cells.

Oleanolic acid (OA) and its several derivatives possess chemopreventive and chemotherapeutic functions against a series of cancer types. Many chemotherapeutic compounds are effective in improving the quality of life and prolonging the survival of patients with gastric cancer, therefore progress in the treatment of gastric cancer, especially the anticancer effects of OA derivatives must be achieved. The inhibitory effect of SZC017, a newly synthesized derivative of OA, on cell viability was determined by MTT assay. Furthermore, flow cytometry, transmission electron microscopy, and western blot analysis revealed that the inhibition of cell viability by OA was mediated by triggering the intrinsic apoptosis of gastric cancer cells, and inducing S phase arrest of SGC7901 cells. Mechanistically, SZC017 was effective against gastric cancer cells via inhibiting Akt/NF­κB signaling and topoisomerase I and IIα proteins. Taken together, our data indicate that SZC017 may be a potential chemotherapeutic agent against gastric cancer cells.

mir-24 activity propagates stress-induced senescence by down regulating DNA topoisomerase 1.

MicroRNAs (miRNAs) are a group of small non-coding executor RNAs. Their function as key modulators of cellular senescence has been widely recognized recently. By cross-comparing several human aging models we previously identified dozens of miRNAs being differentially regulated during aging. Here the functions of two miRNAs, mir-24 and mir-424, were investigated in an oxidative stress-induced fibroblast premature senescence model. Using pre-miRNA precursors, miRNAs were overexpressed in cells undergoing premature senescence induced by oxidative stress. More senescent cells were observed in mir-24 transfected cells. p53 was upregulated in mir-24 overexpressing cells, but downregulated in mir-424 overexpressing cells. DNA topoisomerase I (TOP1), an enzyme controlling DNA topology, was identified as a target of mir-24, whose expression was induced by oxidative stress. Knocking down TOP1 induced cellular senescence. These results suggest that mir-24 activity propagates stress-induced senescence by down regulating TOP1.

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines.

BACKGROUND: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. METHODS: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. RESULTS: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. CONCLUSIONS: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.

Autoregulation of topoisomerase I expression by supercoiling sensitive transcription.

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis.

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right­ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right­sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.

ß-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells.

OBJECTIVE: To investigate the effects of ß-Elemene (ß-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. METHODS: After treatment with ß-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of ß-ELE on DNA breaks. RESULTS: ß-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. ß-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. ß-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. CONCLUSION: ß-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

Effects of cinobufacini injection on cell proliferation and the expression of topoisomerases in human HepG-2 hepatocellular carcinoma cells.

The present study aimed to investigate the effects of cinobufacini injection on the proliferation and expression of topoisomerases in human HepG-2 hepatocarcinoma cells. The cells were divided into a control group and an experimental group, in which 0.105, 0.21, 0.42 mg/l cinobufacini was injected. Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay, levels of apoptosis were detected using annexin V/propidium iodide staining and cell cycles were analyzed using flow cytometric analysis. The mRNA and protein expression levels of topoisomerase (TOPO) I and TOPO II were determined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Cinobufacini injection significantly inhibited the proliferation of the HepG-2 cells (P<0.05), induced apoptosis (P<0.05) in a dose- and time-dependent manner, induced tumor cell arrest at the S phase in a dose-dependent manner, and downregulated the mRNA and protein expression levels of TOPO I and TOPO II (P<0.05) in a dose-dependent manner. Therefore, cinobufacini was found to inhibit human HepG-2 hepatocellular carcinoma cell proliferation, and downregulation of the expression levels of TOPO I and TOPO II may contribute to the effect on proliferation observed in the Hep­G2 cells following cinobbufacini injection.

Diagnostic criteria of systemic sclerosis.

Systemic sclerosis (SSc) is a multisystem disease characterized by vascular abnormalities, immune system activation manifested by SSc-specific autoantibodies and disturbances in fibroblast function. The clinical manifestations are highly heterogeneous and commonly include skin thickening, Raynaud's phenomenon, digital ulcers, gastroesophageal reflux disease, interstitial lung disease and cardiac diastolic dysfunction. The diagnosis of SSc in a patient with typical end-organ disease is relatively straight-forward, but is unsatisfactory because it implies that the diagnosis is delayed until irreversible tissue damage is present. Diagnostic criteria are generally designed to facilitate the clinical process and to allow early institution of therapy to relieve symptoms and possibly prevent irreversible damage. Several attempts at defining diagnostic criteria for SSc have been made in the past. Raynaud's phenomenon, SSc-specific autoantibodies and nailfold capillary abnormalities are among the most promising items likely to be retained in a final set of diagnostic criteria. The EULAR Scleroderma Trial and Research group (EUSTAR) is currently in the process of prospectively validating a set of diagnostic criteria for the very early diagnosis of SSc and results are expected in 2015.

Cinnamaldehyde/chemotherapeutic agents interaction and drug-metabolizing genes in colorectal cancer.

Cinnamaldehyde is an active monomer isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which is known to possess marked antitumor effects in vitro and in vivo. The aim of the present study was to examine the potential advantages of using cinnamaldehyde in combination with chemotherapeutic agents commonly used in colorectal carcinoma (CRC) therapy, as well as to investigate the effect of cinnamaldehyde on chemotherapeutic-associated gene expression. The synergistic interaction of cinnamaldehyde and chemotherapeutic agents on human CRC HT-29 and LoVo cells was evaluated using the combination index (CI) method. The double staining with Annexin V conjugated to fluorescein-isothiocyanate and phosphatidylserine was employed for apoptosis detection. The expression of drug-metabolizing genes, including excision repair cross­complementing 1 (ERCC1), orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), breast cancer susceptibility gene 1 (BRCA1) and topoisomerase 1 (TOPO1), all in HT-29 and LoVo cells, with or without the addition of cinnamaldehyde, was examined by quantitative polymerase chain reaction (PCR). Cinnamaldehyde had a synergistic effect on the chemotherapeutic agents cytotoxicity in HT-29 and LoVo cells. In addition, cinnamaldehyde suppressed BRCA1, TOPO1, ERCC1 and TS mRNA expression, except for OPRT expression, which was markedly upregulated. Our findings indicate that cinnamaldehyde appears to be a promising candidate as an adjuvant in combination therapy with 5-fluorouracil (5-FU) and oxaliplatin (OXA), two chemotherapeutic agents used in CRC treatment. The possible mechanisms of its action may involve the regulation of drug­metabolizing genes.

Tirapazamine sensitizes hepatocellular carcinoma cells to topoisomerase I inhibitors via cooperative modulation of hypoxia-inducible factor-1α.

Topoisomerase I inhibitors are a class of anticancer drugs with a broad spectrum of clinical activity. However, they have limited efficacy in hepatocellular cancer. Here, we present in vitro and in vivo evidence that the extremely high level of hypoxia-inducible factor-1α (HIF-1α) in hepatocellular carcinoma is intimately correlated with resistance to topoisomerase I inhibitors. In a previous study conducted by our group, we found that tirapazamine could downregulate HIF-1α expression by decreasing HIF-1α protein synthesis. Therefore, we hypothesized that combining tirapazamine with topoisomerase I inhibitors may overcome the chemoresistance. In this study, we investigated that in combination with tirapazamine, topoisomerase I inhibitors exhibited synergistic cytotoxicity and induced significant apoptosis in several hepatocellular carcinoma cell lines. The enhanced apoptosis induced by tirapazamine plus SN-38 (the active metabolite of irinotecan) was accompanied by increased mitochondrial depolarization and caspase pathway activation. The combination treatment dramatically inhibited the accumulation of HIF-1α protein, decreased the HIF-1α transcriptional activation, and impaired the phosphorylation of proteins involved in the homologous recombination repair pathway, ultimately resulting in the synergism of these two drugs. Moreover, the increased anticancer efficacy of tirapazamine combined with irinotecan was further validated in a human liver cancer Bel-7402 xenograft mouse model. Taken together, our data show for the first time that HIF-1α is strongly correlated with resistance to topoisomerase I inhibitors in hepatocellular carcinoma. These results suggest that HIF-1α is a promising target and provide a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and tirapazamine in hepatocellular cancers.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico.

BACKGROUND: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear. METHODS: The three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation. RESULTS: The three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine. CONCLUSIONS: These results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Alpha, beta-unsaturated lactones 2-furanone and 2-pyrone induce cellular DNA damage, formation of topoisomerase I- and II-DNA complexes and cancer cell death.

The alpha, beta-unsaturated lactones 2-furanone and 2-pyrone are part of the chemical structure of a variety of naturally occurring compounds (e.g., cardenolides, bufadienolides, acetogenins, coumarins, and food-flavoring furanones), some of which have shown anticancer activity and/or DNA damaging effects. Here we report that 2-furanone and 2-pyrone induce cellular DNA damage (assessed by the comet assay and the gamma-H2AX focus assay) and the formation of topoisomerase I- and topoisomerase II-DNA complexes in cells (visualized and quantified in situ by the TARDIS assay). Cells mutated in BRCA2 (deficient in homologous recombination repair) were significantly hypersensitive to the cytotoxic activity of 2-pyrone, therefore suggesting that BRCA2 plays an important role in the repair of DNA damage induced by this lactone. Both lactones were cytotoxic in A549 lung cancer cells at lower concentrations than in MRC5 non-malignant lung fibroblasts. The possible involvement of 2-furanone and 2-pyrone in the anticancer and DNA-damaging activities of compounds containing these lactones is discussed.

Phenotypic screening reveals topoisomerase I as a breast cancer stem cell therapeutic target.

Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.

Overexpression of Lewis(y) antigen protects ovarian cancer RMG-1 cells from carboplatin-induced apoptosis by the upregulation of Topo-I and Topo-II ß.

Lewis (y) antigen, a difucosylated oligosaccharide, has been shown to be associated with malignant properties of ovarian carcinomas. In this study, we have investigated the potential role of Lewis (y) antigen, which was stably transfected into ovarian cancer RMG-1 cells, on carboplatin-induced apoptosis. Overexpression of Lewis (y) antigen effectively protected vitronectin-adherent RMG-1 cells from carboplatin-induced apoptosis as assessed by Hoechst 33258 staining and flow cytometry. Treatment with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin ß3 antibody partially abolished the protective effect on apoptosis and markedly inhibited the expression of Topo-II ß in cells overexpressing Lewis (y) antigen (all P < 0.01). Moreover, elevated expression of Topo-I and Topo-II ß was found in Lewis (y) antigen-overexpressing cells (P < 0.01). However, no obvious changes in Topo-II α were observed throughout the study (P > 0.05). Taken together, these data suggest that the overexpression of Lewis (y) antigen confers cell adhesion-mediated drug resistance to apoptosis in ovarian cancer cells by the upregulation of Topo-I and Topo-II ß. Therefore, the inhibition of Lewis (y) antigen may be a novel strategy of cancer chemotherapy.

Exon array analyses across the NCI-60 reveal potential regulation of TOP1 by transcription pausing at guanosine quartets in the first intron.

Because topoisomerase 1 (TOP1) is critical for the relaxation of DNA supercoils and because it is the target for the anticancer activity of camptothecins, we assessed TOP1 transcript levels in the 60 cell line panel (the NCI-60) of the National Cancer Institute's anticancer drug screen. TOP1 expression levels varied over a 5.7-fold range across the NCI-60. HCT116 colon and MCF-7 breast cancer cells were the highest expressers; SK-MEL-28 melanoma and HS578T breast carcinoma cells were the lowest. TOP1 mRNA expression was highly correlated with Top1 protein levels, indicating that TOP1 transcripts could be conveniently used to monitor Top1 protein levels and activity in tissues. Assessment of the TOP1 locus by array comparative genomic hybridization across the NCI-60 showed copy numbers ranging from 1.71 to 4.13 and a statistically significant correlation with TOP1 transcript levels (P < 0.01). Further analyses of TOP1 expression on an exon-specific basis revealed that exon 1 expression was generally higher and less variable than expression of the other exons, suggesting some form of transcriptional pausing regulation between exons 1 and 2. Accordingly, we found the presence of multiple evolutionarily conserved potential G-quadruplex-forming sequences in the first TOP1 intron. Physicochemical tests for actual quadruplex formation by several of those sequences yielded quadruplex formation for two of them and duplex formation for one. The observations reported here suggest the hypothesis that there is a conserved negative transcription regulator within intron 1 of the TOP1 gene associated with a quadruplex-prone region.

Relations between strain and gender dependencies of irinotecan toxicity and UGT1A1, CES2 and TOP1 expressions in mice.

Irinotecan hydrochloride (CPT-11) can display severe toxicities in individual cancer patients. CPT-11 is bio-activated through CES, detoxified through UGT1A1 and inhibits TOP1. CPT-11 toxicity and UGT1A1, CES2 and TOP1 mRNAs and UGT1A1 protein were determined in male and female C57BL/6, B6D2F1 and B6CBAF1, as potential models for tailoring CPT-11 delivery. CPT-11 was administered intravenously (40-90 mg/kg/day for 4 days at 7h after light onset). The relations between dose and lethal toxicity or body weight loss were steep and similar in C57BL/6 (lethality, p=0.001; weight loss, p=0.002) and B6D2F1 (p=0.01; p=0.03, respectively), but weak in B6CBAF1. Females displayed less toxicity than males (p<0.001). Mean mRNA expression of UGT1A1 was highest in B6CBAF1 (p=0.039) and in females (p<0.001). Both CES2 and TOP1 varied according to strain and gender (p<0.001). The three gene expression data explained the most severe toxicity of CPT-11 in male B6D2F1, but displayed inconsistent relations with toxicity in the other groups. Mean UGT1A1 protein expression was highest in males as compared to females, and so by approximately 8-fold in C57BL/6 as compared to B6D2F1 (p<0.0001). Genetic background and gender significantly altered the molecular prediction of irinotecan toxicity by UGT1A1, CES2 and TOP1 mRNA expressions.

Effects of Lewis Y antigen on the gene expression of multiple drug resistance-associated proteins in human ovarian cancer RMG-I-H cells.

The effects of Lewis Y antigen on the gene expression of multiple drug resistance-associated proteins in human ovarian cancer RMG-I-H cells were unclear by now. In this study, we detected the gene expression of multiple drug resistance-associated proteins (MRP) in RMG-I-H cells and RMG-I-H cells treated with anti-Lewis Y monoclonal antibody to investigate the association between Lewis Y antigen and the gene expression of drug resistance-associated proteins. Compared with RMG-I cells, the expression of MRP1, MRP2, protein kinase C-alpha (PKC-alpha), and topoisomerase I (Topo I) mRNAs in RMG-I-H cells were significantly upregulated, while the MDR-1 mRNA was downregulated. Immunochemistry analyses indicated that the in vitro and in vivo expression levels of MDR-1 protein (P-gp) in RMG-I-H cells were significantly higher than those in RMG-I cells. After RMG-I-H cells were treated with anti-Lewis Y monoclonal antibody, the expression levels of MDR-1, MRP1, MRP2, PKC-alpha, and Topo I mRNAs gradually decreased with the prolongation of treatment duration. In contrast, no obvious changes were noted in the expression levels of these mRNAs in the non-treatment group. At 6 h after treatment, the relative levels of MDR-1, MRP1, MRP2, PKC-alpha, and Topo I mRNAs in the antibody treatment group were significantly lower than those in the non-treatment group. In conclusion, Lewis Y antigen is closely associated with regulating the gene expression of multiple drug resistance-associated proteins.

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells.

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.