TOP1 mRNA Research Articles

Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.

Necessity of HuR/ELAVL1 for activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in antibody diversification.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage are the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR/ELAVL1, a protein that binds to AU-rich elements in RNA. HuR-knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR-KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR-KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR-KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR-KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR, and hypermutation in HuR-KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis.

Effect of Pterostilbene, a Natural Derivative of Resveratrol, in the Treatment of Colorectal Cancer through Top1/Tdp1-Mediated DNA Repair Pathway.

Simple SummaryTyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs the stalled Topoisomerase 1 (Top1)-DNA covalent complex. It is conceivable that Tdp1 inhibitors could act synergistically with Top1 inhibitors to enhance the effect of Top1 poisons. This study identified pterostilbene (PTE) and resveratrol (RE) to suppress these two proteins by binding to their active center. PTE and RE could inhibit the proliferation of various colorectal cancer cells and decrease Top1 and Tdp1 contents and mRNA expression in wild-type, constructed Tdp1 overexpressing CL187, Top1- or Tdp1- silenced CL187 cell lines. PTE exhibited better antitumor activity and safety in subcutaneous CL187 transplantion model and orthotopic transplantation model. PTE had no significant inhibitory effect on non-tumor cell proliferation in vitro and would not induce damage to liver, kidney, and other major organs.Topoisomerase 1 (Top1) inhibitor is an effective anticancer drug, but several factors limit its clinical application such as drug inactivation, tyrosyl-DNA phosphodiesterase 1 (Tdp1)-mediated tumor drug resistance, and its toxicity. Our previous study identified pterostilbene (PTE) and resveratrol (RE) to suppress these two proteins by binding to their active center. PTE and RE could inhibit the proliferation of various colorectal cancer cells, induce cell apoptosis, and make cell cycle stay in G2/M phase in vitro. PTE and RE could decrease Top1 and Tdp1 contents and mRNA expression in wild-type, constructed Tdp1 overexpressing CL187, Top1- or Tdp1- silenced CL187 cell lines. PTE exhibited excellent antitumor activity in subcutaneous CL187 transplantation model (TGI = 79.14 ± 2.85%, 200 mg/kg, i.p.) and orthotopic transplantation model (TGI = 76.57 ± 6.34%, 100 mg/kg, i.p.; TGI = 72.79 ± 4.06%, 500 mg/kg, i.g.) without significant toxicity. PTE had no significant inhibitory effect on non-tumor cell proliferation in vitro and would not induce damage to liver, kidney, and other major organs. Overall, PTE and RE can inhibit the activity of Top1 enzyme and inhibit the DNA damage repair pathway mediated by Top1/Tdp1, and can effectively inhibit colorectal cancer development with low toxicity, thus they have great potential to be developed into a new generation of anti-tumor drugs.

Combination of histoculture drug response assay and qPCR as an effective method to screen biomarkers for personalized chemotherapy in esophageal cancer.

Personalized chemotherapy with the use of biomarkers helps to maximize clinical efficiency. Therefore, the present study aimed to identify a potential method for identifying biomarkers in esophageal cancer. A total of 49 freshly resected tumor tissues and 72 paraffin-embedded specimens from patients with esophageal cancer were obtained. mRNA expression levels of ERCC1, BRCA1, TUBB3, FBW7, RRM1, MDM2, TS and TOP1 were measured quantitative reverse transcription polymerase chain reaction (RT-qPCR). In vitro chemosensitivity to cisplatin, docetaxel, gemcitabine, etoposide, fluorouracil and irinotecan were tested using histoculture drug response assay (HDRA). BRCA1 mRNA levels were positively correlated with resistance to cisplatin (P=0.027) and sensitivity to docetaxel (P=0.002). TS mRNA levels were inversely correlated with fluorouracil sensitivity (P=0.044), and TOP1 mRNA expression was positively correlated with irinotecan sensitivity (P=0.008). In addition, high BRCA1 mRNA levels correlated with decreased median overall survival (mOS; P<0.001) and response rate (RR; P=0.002) in cisplatin-fluorouracil chemotherapy group and also correlated with increased mOS (P<0.001) and RR (P=0.023) in docetaxel-fluorouracil chemotherapy group. Overall, these results suggested that HDRA combined with RT-qPCR may serve as an effective method for screening biomarkers in personalized chemotherapy for esophageal cancer.

ABCG2 and TOP-1 mRNA expression as predictive biomarkers for adjuvant FOLFIRI treatment in stage III colon cancer patients: Results from the PETAAC-3 prospective randomized clinical trial.

11594 Background: FOLFIRI as adjuvant treatment in primary colon cancer was previously tested in two pivotal prospective randomized clinical trials (PETACC-3 and CALGB 89803), both of which failed to demonstrate significant beneficial effects when adding irinotecan to 5FU. As a consequence, FOLFIRI is presently not used as adjuvant treatment for colon cancer. Methods: The study included 580 patients with mRNA expression data performed on tumor samples (FFPE) from stage III colon cancer patients enrolled in the PETACC-3 study, which randomized the patients to 5FU plus Leucovorin +/- irinotecan. Primary end-points were recurrence-free survival (RFS) and overall survival (OS). Median ABCG2 and the 75 percentile TOP-1 mRNA expression data were used to allocate the patients into one of two groups: One with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (n = 167) and another group including all other combinations of these two genes. Kaplan Meier curves and Cox proportional hazards model were used to visualize differences between groups and calculate p-values (log-rank test). Results: The survival statistics showed a significant difference for both RFS (HR: 0.63 (0.44-0.92); p = 0.017) and OS (HR: 0.6 (0.39-0.93); p = 0.021) between the two groups when the patients received FOLFIRI. In contrast, no significant differences were observed between the groups when patients received 5FU and Leucovorin alone (p-values: RFS: 0.58; OS: 0.75). Conclusions: We here show that the combination of two independent gene expression abundance with a strong association to irinotecan treatment (high ABCG2 drug efflux pump and low TOP-1, the latter being the target for irinotecan) identified a group of stage III colon cancer patients who will not benefit from FOLFIRI adjuvant treatment while patients with other combinations of expression of these two genes appear to significantly benefit from adjuvant FOLFIRI treatment. The lack of a similar effect in patients receiving treatment with 5FU and Leucovorin only, points to a predictive value of ABCG2 and TOP-1 measurements.

ABCG2 and TOP1 mRNA expression as predictive biomarkers for adjuvant FOLFIRI treatment in stage III colon cancer patients: Results from the PETAAC-3 prospective randomized clinical trial.

591 Background: FOLFIRI as adjuvant treatment in primary colon cancer was previously tested in two pivotal prospective randomized clinical trials (PETACC-3 and CALGB 89803), both of which failed to demonstrate significant beneficial effects when adding irinotecan to 5FU. As a consequence, FOLFIRI is presently not used as adjuvant treatment for colon cancer. Methods: The study included 580 patients with mRNA expression data performed on tumor samples (FFPE) from stage III colon cancer patients enrolled in the PETACC-3 study, which randomized the patients to 5FU plus Leucovorin +/- irinotecan. Primary end-points were disease free survival (DFS) and overall survival (OS). Median ABCG2 and the 75 percentile TOP-1 mRNA expression data were used to allocate the patients into one of two groups: One with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (n = 167) and another group including all other combinations of these two genes. Kaplan Meier curves and Cox proportional hazards model were used to visualize differences between groups and calculate p-values (log-rank test). Results: The survival statistics showed a significant difference for both RFS (HR: 0.63 (0.44-0.92); p = 0.017) and OS (HR: 0.6 (0.39-0.93); p = 0.021) between the two groups when the patients received FOLFIRI. In contrast, no significant differences were observed between the groups when patients received 5FU and Leucovorin alone (RFS: 0.58; OS: 0.75). Conclusions: We here show that the combination of two independent gene expression abundance with a strong association to irinotecan treatment (high ABCG2 drug efflux pump and low TOP-1, which is the target for irinotecan) identified a group of stage III colon cancer patients who will not benefit from FOLFIRI adjuvant treatment while patients with other combinations of expression of these two genes appear to significantly benefit from adjuvant FOLFIRI treatment. The lack of a similar effect in patients receiving treatment with 5FU and Leucovorin only, points to a predictive value of ABCG2 and TOP-1 measurements.

Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1—CPT, actinomycin D (ActD) and β-lapachone (β-Lap)—on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system.

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

Prospective-retrospective assessment of the TOP1 gene copy number and TOP1 mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan. TOP1 copy number status was analyzed by fluorescence in situ hybridization (FISH) using a TOP1/CEN20 dual-probe combination. TOP1 mRNA data were available from previous analyses. TOP1 FISH and follow-up data were obtained from 534 patients. TOP1 gain was identified in 27% using a single-probe enumeration strategy (≥4 TOP1 signals per cell) and in 31% when defined by a TOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent on TOP1 FISH status.TOP1 mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized by TOP1 mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment by TOP1 mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasing TOP1 mRNA values appeared to be associated with increasing benefit of irinotecan. In contrast to the TOP1 copy number, a trend was demonstrated for a predictive property of TOP1 mRNA expression. On the basis of TOP1 mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.

Bromodomain and hedgehog pathway targets in small cell lung cancer

Small cell lung cancer (SCLC) is an extremely aggressive cancer that frequently recurs. Twenty-three human SCLC lines were selected representing varied Myc status. Gene expression of lung cancer, stem-like, hedgehog pathway, and notch pathway genes were determined by RT2-PCR array and Exon 1.0 ST array. Etoposide and topotecan concentration response was examined. The IC50's for etoposide and topotecan ranged over nearly 3 logs upon 96 hrs exposure to the drugs. Myc status, TOP2A, TOP2B and TOP1 mRNA expression or topoisomerase 1 and topoisomerase 2 protein did not account for the range in the sensitivity to the drugs. γ-secretase inhibitors, RO429097 and PF-03084014, had little activity in the SCLC lines over ranges covering the clinical Cmax concentrations. MYC amplified lines tended to be more sensitive to the bromodomain inhibitor JQ1. The Smo antagonists, erismodegib and vismodegib and the Gli antagonists, HPI1 and SEN-450 had a trend toward greater sensitivity of the MYC amplified line. Recurrent SCLC is among the most recalcitrant cancers and drug development efforts in this cancer are a high priority.

TOP1 gene copy numbers are increased in cancers of the bile duct and pancreas

Background. Bile duct and pancreatic cancer (PC) have poor prognoses and treatment options for inoperable patients are scarce. In order to improve outcome for these patients, there is an urgent need for biomarkers predictive of treatment effect. Irinotecan is a topoisomerase 1 (Top1) poison. Top1 protein, TOP1 gene copy number and mRNA expression, respectively, have been proposed as predictive biomarkers of response to irinotecan in other cancers. Here we investigate the occurrence of TOP1 gene aberrations in cancers of the bile ducts and pancreas. Material and methods. TOP1 and centromere 20 (CEN-20) numbers were investigated by fluorescence in situ hybridization analyses in tumor tissue from 226 patients. The frequencies of aberration in the TOP1 gene copy number, the CEN-20 copy number and the TOP1/CEN-20 ratio were analyzed. As TOP1 is located on chromosome 20, the CEN-20 probe was included to distinguish between chromosomal and gene amplifications. Results. In PC, 29.8% had an increased TOP1 copy number (≥3.5n gene copies per cell) and 10.8% had a TOP1/CEN-20 ratio >1.5. In bile duct cancer, 12.8 % had an increased TOP1 copy number and 6.4% had a TOP1/CEN-20 ratio >1.5. Neither the TOP1 copy number nor the TOP1/CEN-20 ratios could predict overall survival. Conclusion. We here report that a substantial number of patients with bile duct or PC have increased TOP1 copy number and increased TOP1/CEN-20 ratio making further analyses on the association between TOP1 gene copy number and irinotecan efficacy clinically relevant.

Circadian Rhythm of Anti-Cancer Drug Target Topoisomerase I As a Proposal Model for Chronotherapy In Vitro

Many behavioral processes are under circadian regulation in almost all organisms from bacteria to mammals. Although substantial evidences support the idea that circadian clock could modulate the efficacy of anticancer therapy, the mechanism(s) has not been clear. Here we demonstrated that expression of topoisomerase I (top1) mRNA and its promoter activity underwent circadian oscillation after serum-rich medium treatment in HCT 116 cells. Moreover, the formation of cleavage complex composed of top1 and top1 poison, camptothecin (CPT), detected by nicking assay was also oscillated. Colony formation assay indicated that cytotoxicity of 8h-CPT treatment was significantly higher at 4-h after serum-shock than at 20-h (p = 0.0289). In addition, dexamethasone treatment also induced circadian rhythm of top1 in HCT 116 cells. These results raise the possibility that the circadian rhythm of top1, which would be induced by some types of drugs such as dexamethasone, could be considered for chemotherapy in clinic.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Abstract C149: Exon array analyses across the NCI-60 reveals potential regulation of TOP1 by transcription pausing at guanosine quartets in the first intron

Abstract Because Topoisomerase I (TOP1) is critical for the relaxation of DNA supercoils and because it is the target for 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&amp;lt;0.01). Further analyses of TOP1 expression on an exon-specific basis revealed that exon 1 expression appeared to be generally higher, and less variable than the other exons, suggesting some form of transcriptional pausing regulation between exons one and two. Accordingly, we found the presence of multiple evolutionarilly-conserved potential G-quadruplex-forming sequences in the first TOP1 intron. Physico-chemical 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, and the regulator is a quadruplex-prone region. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C149.

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 7 h 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 ∼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.

Topoisomerase I levels in the NCI-60 cancer cell line panel determined by validated ELISA and microarray analysis and correlation with indenoisoquinoline sensitivity

Topoisomerase I (Top1) is a proven target for cancer therapeutics, and the level of Top1 in tumors has been used as a biomarker for chemotherapeutic efficacy. In this study, we report the development and validation of a two-site enzyme chemiluminescent immunoassay for Top1, which we used to measure Top1 levels in the NCI-60 cancer cell line panel. Top1 levels ranged from 0.9 to 9.8 ng/mL/microg protein extract in these cell lines. Levels varied both within and between cancer types but were generally highest in colon cancer and leukemia cell lines and lowest in central nervous system and renal cancer cell lines. Top1 mRNA levels in the NCI-60 cell lines were also measured by microarray; mRNA values generally showed a good correlation with protein levels (Pearson correlation = 0.8). When these expression levels were compared with the activity of the indenoisoquinoline class of Top1 inhibitors across the NCI-60 cell panel, low levels of Top1 were associated with increased resistance to these drugs. The results of our studies indicate that our Top1 assay can be used to quantify Top1 levels in untreated cells as well as cells treated with Top1 inhibitors and that the assay has the potential to be adapted for use in predicting clinical response to Top1-active antineoplastic agents.

Analysis of topoisomerase (TOP) expression in peripheral blood mononuclear cell (PBMCs) from patients (PTS) undergoing chemotherapy (CHT) for solid tumors( ST)

2124 Background: Clinical experience demonstrated the efficacy of combined drug regimens, including inhibitors of DNA replication and anti-TOP. Aim of this study was the time-course evaluation of TOP mRNA expression in PBMCs from PTS undergoing CHT for histologically proven ST in order to optimise combination regimens including TOP inhibitors. Methods: We analysed TOP1, TOP2A, TOP2B mRNA levels in PBMCs from 33 PTS (18 M and 15 F, median age 63) by quantitative real-time RT-PCR. Measurements were performed at 0 h, before the beginning of the first CHT dose administration, and at +2, +3, +6 and +24 h. Drugs included gemcitabine, oxaliplatin, 5-fluorouracile and methotrexate, either alone or in combination. Results: 28 PTS showed a significant increase of TOP expression at 2–3 h from the beginning of treatment with a subsequent restoration of baseline values at 6 h. In particular, TOP1 mRNA levels increased from 120% to 1,400% of baseline values, TOP2A increased 200%–730% from baseline and TOP2B increased from 130%–12,000% of baseline. Among the variety of drugs tested, 5-fluorouracile (in monotherapy or in association with oxaliplatin or methotrexate) and gemcitabine were the most effective in inducing TOP expression. Conclusions: we demonstrated that anti-tumour drugs acting on cell replication and/or metabolism induce TOP1, TOP2A, TOP2B expression with a pick at 2–3 h from treatment. These results are of interest in the designing combined chemotherapy schedules including topoisomerase inhibitors. No significant financial relationships to disclose.

Developmental Regulation of DNA-Topoisomerases during Drosophila Embryogenesis

Type I and type II DNA-topoisomerases are essential enzymes that mediate replication, transcription, recombination, and mitosis in multicellular eukaryotes but the extent of their interchange for specific reactions in vivo is controversial. Expression patterns for topoisomerase I and topoisomerase II during the embryogenesis of Drosophila melanogaster were compared with patterns of DNA replication and expression of the histone genes. In late oogenesis the maternally supplied top2 mRNA was evenly distributed throughout the egg with elevated levels at the posterior tip, a pattern that is maintained in syncytial blastoderm embryos. During gastrulation, top2 mRNA became differentially localized only to regions of DNA replication, including new expression in the gonads preceding mitosis/meiosis. Significantly higher levels of top2 mRNA were found in mitotic compared to endoreplicating tissues. The total histone mRNA was exclusively associated with DNA replication but, in contrast to top2 mRNA, mitotic and endoreplicating cells contained similar expression levels with no expression in the gonads. Striking differences exist between the distribution of the top2 mRNA and topoisomerase II protein. The protein localizes to all evolving nuclei where it persists throughout embryogenesis. A high level of top1 mRNA transcript was present without differential tissue distribution throughout embryogenesis.

Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma.

In previous studies, we established two camptothecin (CPT)‐resistant sublines, HT‐29/CPT and St‐4/CPT, from the human colon cancer cell line HT‐29 and the human stomach cancer cell line St‐4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight‐fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT‐29/CPT. The longer mRNA (4.0 kb) is the wild‐type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58–729 or 59–730) that caused the in‐frame deletion of amino acids 20–243 of human topo I. The deleted region is identical to exons 3–9 of the TOP1 gene. The expression level of the 3.3‐kb mRNA was similar to that of the wild‐type mRNA in HT‐29/CPT. St‐4/CPT expressed only the wild‐type TOP1 mRNA in lesser amounts than did St‐4. Mouse NIH3T3 cells transfected with the wild‐type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT‐sensitive topo I in the resistant cells.