Topoisomerase 2B Research Articles

Abstract 12348: Targeted Degradation of Topoisomerase 2b by Dexrazoxane for Prevention of Doxorubicin-Induced Cardiotoxicity: Dose and Time Course Study in Human

Introduction: Doxorubicin is known to induce cardiotoxicity through topoisomerase 2b (Top2b), whereas doxorubicin poisons topoisomerase 2a (Top2a) to kill cancer cells. When Top2b was genetically deleted in the cardiomyocytes of adult mice, doxorubicin-induced cardiomyopathy was prevented. Pharmacological degradation of Top2b by dexrazoxane can also prevent doxorubicin-induced cardiotoxicity in mice. However, it is not known whether dexrazoxane can induce degradation of Top2b in human. Hypothesis: Targeted degradation of Top2b by dexrazoxane can be achieved in humans. Methods: Top2b protein level in peripheral blood mononuclear cells (PBMC) were assessed serially after intravenous dexrazoxane infusion in human volunteers. The effectiveness of dexrazoxane on Top2b degradation and the time course of Top2b degradation were determined. Blood was drawn before dexrazoxane infusion, hourly after infusion up to 12 hours, at 24 hours, and at 48 hours. PBMCs were isolated using SepMate tube and fractionated into nuclear and cytosolic fractions. The nuclear fractions were used in Western blot analysis. Immunoblotting was carried out with rabbit anti-Top2b, anti-Top2a, or anti-Lamin B1 antibodies, followed by Peroxidase AffiniPure goat anti-rabbit IgG. The ratio of the intensity of the Top2b and Lamin B1 bands were normalized by setting the pre-ratio to 1. Results: One hour after dexrazoxane infusion, Top2b level in leukocytes was reduced to 14%-21% of baseline. Dexrazoxane-induced degradation of Top2b persisted for up to 12 hours and returned to baseline 24 hours after dexrazoxane infusion. Importantly, Top2a level did not vary significantly following dexrazoxane infusion. There were no serious adverse reactions in all participants. Conclusions: Human PBMC can be used as a surrogate to follow Top2b degradation. Degradation of Top2b can be achieved with dexrazoxane dose as low as 100 mg/M 2 (one-fifth of the current FDA recommended dose when used in conjunction with doxorubicin). Thus, early dexrazoxane followed by delayed-administration of doxorubicin can be studied as a new strategy to prevent doxorubicin-induced cardiotoxicity by depleting Top2b. Moreover, dexrazoxane did not affect Top2a, thus preserving doxorubicin’s efficacy.

The Bisdioxopiperazine ICRF-193 Attenuates LPS-induced IL-1β Secretion by Macrophages

Inhibiting pathological secretion of Interleukin-1β has shown beneficial effects in disease models and in the clinic and thus there is interest in finding inhibitors that can reduce its release from macrophages in response to their activation by foreign pathogens. We used an in vitro human macrophage model to investigate whether ICRF-193, a Topoisomerase II inhibitor could modulate IL1B mRNA expression and IL-1β secretion. These macrophage-like cells readily secrete IL-1β in response to Lipopolysaccharide (LPS). Upon exposure to a non-toxic dose of ICRF-193, IL-1β secretion was diminished by ~ 40%; however, level of transcription of IL1B was unaffected. We show that there was no Topoisomerase 2B (TOP2B) binding to several IL1B gene sites, which may explain why ICRF-193 does not alter IL1B mRNA levels. Hence, we show for the first time that ICRF-193 can reduce IL-1β secretion. Its low cost and the development of water-soluble prodrugs of ICRF-193 warrants its further investigation in the modulation of pathological secretion of this cytokine for the treatment of inflammatory disorders. (165 words).

A Review on Anthracycline Induced Cardiotoxicity- A Mechanism-based Approach

Doxorubicin has become one of the most effective chemotherapeutic agents, but its use was complicated by the development of heart failure. Proposed mechanisms for its antitumor effects included intercalation into DNA that caused the prevention of micro molecule synthesis, DNA cross-linkage and binding, DNA damage due to topoisomerase 2b suppression, reactive oxygen species production, and induction of apoptosis. Several drugs such as ACE inhibitors or the angiotensin receptor blockers, beta-blockers and the CHF therapy are used for the treatment processes. The present brief review of the literature, focuses on literature based on the mechanism of anthracycline-induced cardiotoxicity.

Abstract 9710: Prevention of Doxorubicin-Induced Cardiotoxicity Through Targeted Degradation of Topoisomerase 2b

Introduction: Doxorubicin-induced cardiotoxicity is a serious complication of cancer therapy recognized 40 years ago and remains to be a major clinical challenge today. Doxorubicin poisons topoisomerase 2a to kill cancer cells, whereas doxorubicin induces cardiotoxicity through topoisomerase 2b. When topoisomerase 2b was genetically deleted in the cardiomyocytes of adult mice, doxorubicin-induced cardiomyopathy can be prevented. Hypothesis: Pharmacological depletion of topoisomerase 2b in cardiac tissue could prevent doxorubicin-induced cardiomyopathy. Methods: Cell lines were treated with dexrazoxane (100 μM) for different time intervals with or without the proteasome inhibitor MG132 (10 μM). Cell lysates were immunoblotted with anti-topoisomerase 2a or 2b antibodies or anti-actin. Mice were injected with dexrazoxane (100 mg/kg IP) and cardiac tissues were analyzed for expression of topoisomerase 2b over time using western blot analysis. To study the effect of dexrazoxane pre-treatment on doxorubicin-induced chronic heart failure, C57BL/6 mice (n=7) were pre-treated with dexrazoxane (100 mg/kg IP) 8 hours before doxorubicin (5 mg/kg IP). Single injection of dexrazoxane (n=6), doxorubicin (n=8), or saline control (n=6) were also administered to three different cohorts of mice. This protocol was repeated weekly for 5 weeks. Pre-treatment and post-treatment cardiac MRI were obtained to evaluate the change in left ventricular ejection fraction. Results: Dexrazoxane induced a ubiquitin-dependent degradation of topoisomerase 2b, but not 2a in cell lines. In dexrazoxane-treated mice, topoisomerase 2b was not detectable 8 hours after dexrazoxane administration, but gradually returned to normal levels by 48 hours. Dexrazoxane pre-treatment 8 hours before doxorubicin prevented doxorubicin-induced reduction in ejection fraction as determined by cardiac MRI in a chronic heart failure model. Conclusions: Targeted degradation of topoisomerase 2b by dexrazoxane prevented doxorubicin-induced cardiotoxicity. Through selective degradation of topoisomerase 2b, but not 2a, doxorubicin's tumoricidal activity is preserved. This preventive strategy will be tested in a prospective clinical trial in breast cancer patients.

Novel localizations and interactions of intercellular bridge proteins revealed by proteomic profiling†.

Intercellular bridges (ICBs) connecting germ cells are essential for spermatogenesis, and their deletion causes male infertility. However, the functions and component factors of ICBs are still unknown. We previously identified novel ICB-associated proteins by proteomics analysis using ICB enrichment. Here, we performed immunoprecipitation-proteomics analyses using antibodies specific to known ICB proteins MKLP1, RBM44, and ectoplasmic specialization-associated protein KIAA1210 and predicted protein complexes in the ICB cores. KIAA1210, its binding protein topoisomerase2B (TOP2B), and tight junction protein ZO1 were identified as novel ICB proteins. On the other hand, as well as KIAA1210 and TOP2B, MKLP1 and RBM44, but not TEX14, were localized at the XY body of spermatocytes, suggesting that there is a relationship between ICB proteins and meiotic chromosomes. Moreover, small RNAs interacted with an ICB protein complex that included KIAA1210, RBM44, and MKLP1. These results indicate dynamic movements of ICB proteins and suggest that ICB proteins could be involved not only in the communication between germ cells but also in their epigenetic regulation. Our results provide a novel perspective on the function of ICBs and could be helpful in revealing the biological function of the ICB.

Neonatal murine engineered cardiac tissue toxicology model: Impact of dexrazoxane on doxorubicin induced injury

Doxorubicin (DOX) induced cardiotoxicity is a life-threatening side effect of chemotherapy and decreased cardiac function can present years after treatment. Despite the investigation of a broad range of pharmacologic interventions, to date the only drug shown to reduce DOX-related cardiotoxicity in preclinical studies and limited clinical trials is the iron chelating agent, dexrazoxane (DRZ), although the mechanisms responsible for DRZ mediated protection from DOX related cardiotoxicity remain unclear. Engineered cardiac tissues (ECTs) can be used for tissue repair strategies and as in vitro surrogate models to test cardiac toxicities and preventative countermeasures. Neonatal murine ECTs display cardiotoxicity in response to the environmental toxin, cadmium, and reduced cadmium toxicity with Zinc co-treatment, in part via the induction of the anti-oxidant Metallothionein (MT). We adapted our in vitro ECT model to determine the feasibility of using the ECT approach to investigate DOX-related cardiac injury and DRZ prevention. We found: (1) DOX induced dose and time dependent cell death in ECTs; (2) Zinc did not show protection from DOX cardiotoxicity; (3) MT overexpression induced by Zinc, low dose Cd pretreatment, or MT-overexpression (MT-TG) did not reduce ECT DOX cardiotoxicity; (4) DRZ reduced ECT DOX induced cell death; and (5) The mechanism of DRZ ECT protection from DOX cardiotoxicity was topoisomerase 2B (TOP2B) inhibition rather than reduced reactive oxygen species. Our data support the feasibility of ECTs as an in vitro platform technology for the investigation of drug induced cardiotoxicities including the role of TOP2B in DOX toxicity and DRZ mediated DOX toxicity prevention.

The cardioprotective effect of dexrazoxane (Cardioxane) is consistent with sequestration of poly(ADP-ribose) by self-assembly and not depletion of topoisomerase 2B.

Following systematic scrutiny of the evidence in support of the hypothesis that the cardioprotective mechanism of action of dexrazoxane is mediated by a ‘depletion’ or ‘downregulation’ of Top2β protein levels in heart tissue, the author concludes that this hypothesis is untenable. In seeking to understand how dexrazoxane protects the heart, the outcomes of a customised association rule learning algorithm incorporating the use of antecedent surrogate variables (CEME, 2017 McCormack Pharma) reveal a previously unknown relationship between dexrazoxane and poly(ADP-ribose) (PAR) polymer. The author shows how this previously unknown relationship explains both acute and long-term cardioprotection in patients receiving anthracyclines. In addition, as a direct inhibitor of PAR dexrazoxane has access to the epigenome and this offers a new insight into protection by dexrazoxane against doxorubicin-induced late-onset damage [McCormack K, manuscript in preparation]. Notably, through this review article, the author illustrates the practical application of probing natural language text using an association rule learning algorithm for the discovery of new and interesting associations that, otherwise, would remain lost. Historically, the use of CEME enabled the first report of the capacity of a small molecule to catalyse the hybrid self-assembly of a nucleic acid biopolymer via canonical and non-canonical, non-covalent interactions analogous to Watson Crick and Hoogsteen base pairing, respectively.

Genome Organization Drives Chromosome Fragility

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.

Molecular Mechanisms of Cardiotoxicity: A Review on Major Side-effect of Doxorubicin

Doxorubicin is among the most powerful drugs used for the treatment of both adult and child cancers. Doxorubicin is a major cause of chemotherapy-induced cardiotoxicity that is a restricting factor for an optimum dose of the drug for treatment of the cancer patients. Many studies have explored pathophysiology and mechanisms of doxorubicin-induced cardiotoxicity. Cellular and animal experiments proposed that doxorubicin-induced cardiotoxicity mechanism is multifactorial. Oxidative stress has been considered as the primary cause of cardiotoxicity. Although there is no effective treatment for doxorubicin-induced cardiotoxicity currently but many investigations are being done to discover prevention treatments whereas no specific treatment has been approved. Studies have shown that reactive oxygen species and topoisomerase 2b are molecular targets for cardioprotection. Therapeutic imaging methods and cardio-biomarkers may be helpful in the improvement of rapid detection of cardiac damage. In this review, effects of doxorubicin on DNA damage, free radical generation, mitochondrial damage, cell death, and other parameters have been studied.

Developmental Epigenetic Programming of Adult Germ Cell Death Disease: Polycomb Protein EZH2–miR-101 Pathway

The Developmental Origin of Health and Disease refers to the concept that early exposure to toxicants or nutritional imbalances during perinatal life induces changes that enhance the risk of developing noncommunicable diseases in adulthood. Patients/materials & methods: An experimental model with an adult chronic germ cell death phenotype resulting from exposure to a xenoestrogen was used. A reciprocal negative feedback loop involving decreased EZH2 protein level and increased miR-101 expression was identified. In vitro and in vivo knockdown of EZH2 induced an apoptotic process in germ cells through increased levels of apoptotic factors (BIM and BAD) and DNA repair alteration via topoisomerase 2B deregulation. The increased miR-101 levels were observed in the animal blood, meaning that miR-101 may be a part of a circulating mark of germ cell death. miR-101-EZH2 pathway deregulation could represent a novel pathophysiological epigenetic basis for adult germ cell disease with environmental and developmental origins.

Abstract 3848: Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia

Abstract Resistance to chemotherapy is a major challenge in the field of leukemia. Since, cancer cells can acquire resistance to DNA damage based therapy by modulating DNA Damage Repair (DDR) pathways, an in-depth understanding of DDR is important for novel drug discoveries. Hence, to understand relation between DDR and resistance we used doxorubicin resistant sub-line (K562-R) of K562 and Mitoxantrone resistant sub-line (HL60/MX) of HL60 as a model system. We show that lethal doses of Mitoxantrone to HL60 and HL60/MX do not induce DNA DSBs (double strand breaks) and accordingly there is no activation of Chk2, although there is equal uptake of drug in both, sensitive and resistant cell types. Since Mitoxantrone produce DSBs by targeting TOP2B, we checked the expression of TOPB2 and found significant decrease in the levels of topoisomerase 2 B (TOP2B). Moreover, negative regulator of TOPB2, mir 21-5P is upregulation in untreated HL60/MX explaining the absence of DSBs in HL-60/MX with Mitoxantrone treatment. Surprisingly, although there are no DSBs induced, we find high levels of DNA-PKcs, MRE11 and Rad50 in HL-60/MX. Furthermore, inhibition of DNA-PKcs re-sensitizes HL-60/MX cells to Mitoxantrone, suggesting a DNA damage independent role for DNA-PK in HL-60/MX cells. Recently DNA-PKcs is shown to play key role in fat metabolism. Indeed we find significant differences in the metabolism of HL60 and HL-60/MX where HL-60 cells use glucose but HL-60/MX cells show reduced glucose and glutamine metabolism and an over expression of fatty acid synthase. However link between DNA-PKcs over expression and differential metabolism in these cells still need to be explored. Interestingly, in contrast to HL60 model, Doxorubicin does induce equal DSBs in K562 and K562-R cells. However K562-R cells hyperactivate ATM and Chk2 to repair their DNA and survive. Importantly, we show that K562 start to show elevated activation of ATM and Chk-2 as early as two rounds of drug treatment. Accordingly, ATM inhibition leads to induction of apoptosis in resistant K562 cells. In conclusion, we show that K562 and HL60 cells use the DNA double strand break repair proteins in DNA damage dependent and independent pathways to acquire resistance to DSB inducing chemo therapeutics. Our data also highlight the importance of considering subtype specific therapeutics in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Sameer Salunkhe, Ekjot Kaur, Ashwin Ramaswamy, Ketaki Patkar, Shilpee Dutt. Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3848. doi:10.1158/1538-7445.AM2015-3848

Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development

Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

Paternal Pronuclei in Mouse Zygotes Created by Injection with Sperm with Fragmented DNA Have Delayed and Incomplete DNA Replication.

We previously reported that when vas deferens sperm are treated with Mn+2 and Ca+2 they cleave their DNA to loop-sized fragments of 25 to 50 kb in a process termed Sperm Chromatin Fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B), followed by irreversible DNA degradation by a nuclease(s). We also reported that when vas deferens SCF spermatozoa were injected into oocytes, the resulting paternal pronuclei did not initiate DNA replication, even though the maternal pronuclei replicated normally. Furthermore, the paternal DNA appeared to be degraded as evidenced by ethidium bromide staining. Here, we followed the fate of the paternal DNA in zygotes created by injection of sperm that had been induced to fragment their DNA (SCF-zygotes). We first tested whether the TUNEL assay could detect the DNA fragmentation in paternal pronuclei of SCF-zygotes. We found that even though we could verify that the sperm DNA had been fragmented to about 25 kb by field-inversion gel electrophoresis, the TUNEL assay was not sensitive enough to identify these breaks. We next tested for H2AFX phosphorylation to determine whether SCF-zygotes recognized the DNA damage. We found that paternal pronuclei resulting from injecting oocytes with SCF spermatozoa stained positively for phoshorylated H2AX at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. In control embryos injected with untreated sperm, neither pronuclei were positive. We then tested our original assertion that paternal pronuclei in SCF-zygotes did not replicate their DNA. In our previous study, we examined embryos at 7 hrs after fertilization, about 2 hrs after DNA synthesis normally initiates, and found that very few paternal pronuclei in SCF-zygotes initiated DNA synthesis. Here, we examined the zygotes at several time points up to 20 hrs after fertilization. We found that SCF-zygotes created after injection of vas deferens sperm with fragmented DNA did replicate paternal DNA, but that the initiation was delayed for up to 10 hrs. Chromosomal analysis of these zygotes demonstrated that in the majority of them paternal chromosomes were abnormal and prematurely condensed, while the maternal pronuclei formed normal chromosomes. A significant number of chromosome breaks were also detected in the paternal complements. These data demonstrate that even though the one-cell embryo recognizes damaged DNA in paternal pronucleus, it still attempts to replicate it, although the replication is much delayed, and may not be complete. This delay may be an attempt by the embryo to repair the DNA breaks before replication is initiated. If so, the premature condensation of paternal chromosomes (PCC) may be the result of the asynchronous initiation of DNA synthesis in the two pronuclei; completed DNA synthesis and initiation of mitosis in the maternal pronucleus causing the incompletely replicated paternal DNA to condense prematurely. Alternatively, the paternal PCC may represent damaged DNA that could not be replicated. In either case, the data demonstrate that sperm DNA damage that is below the level detectable by TUNEL can severely affect early embryo development. This research was supported by NIH grant HD060722.

P5-01-14: Phosphoproteomic Analysis of TIMP-1 Overexpressing MCF-7 Human Breast Cancer Cells Reveals Increased Expression and Phosphorylation of Topoisomerase Proteins.

Abstract Background Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. Lack of TIMP-1 protein either alone (Willemoe et al., Eur J Cancer 2009) or in combination with Topoisomerase 2A gene aberrations has been shown to associate with increased benefit from adjuvant treatment with a Topoisomerase 2 inhibitor (epirubicin containing combination chemotherapy) while this association was not observed in patients treated with a non-Topoisomerase 2 inhibitor combination chemotherapy (Ejlertsen et al., JCO 2010). Aim: To further investigate the molecular mechanisms underlying the association between TIMP-1 and epirubicin sensitivity by quantitative phosphoproteomics. Methods: MCF-7 human breast cancer cells were transfected with pcDNA3.1(Hyg)-TIMP-1. Among 11 single cell clones, two TIMP-1 low expressing and two TIMP-1 high expressing clones were selected. The clones were labeled by SILAC (stable isotope labeling with amino acids in cell culture). Lysates were digested with trypsin and fractionated with SCX followed by subsequent enrichment of phosphopeptides by TiO2-based chromatography and desalting by C18 purification. Total peptides and phosphopeptides were analyzed by tandem mass spectrometry and quantified as described (JV Olsen et al., Science Signaling 2010). Selected proteins were confirmed on Western blots. The sensitivity of the four TIMP-1 cell clones was analyzed by treatment of the cells with the following drugs: The Topoisomerase 2 inhibitor epirubicin (an anthracycline). The Topoisomerase 1 inhibitor SN-38 (the active metabolite of irinotecan, a camptothecin analogue) and the combination of these. A specific Topoisomerase 2B inhibitor (XK 469, a quinoxaline analogue and the DNA crosslinker cisplatin. All experiments were determined with an endpoint MTT assay. Results: The quantitative proteomic analyses confirmed the differences in TIMP-1 levels among the four clones. Several proteins were consistently found to be upregulated and/or had changed phosphorylation levels in the TIMP-1 high cells in two biological replicates. Of particular interest was the observation that the phosphorylation status and protein levels of Topoisomerase-1, -2A and -2B were increased in TIMP-1 high expressing cells compared to TIMP-1 low expressing cells. When the four clones were treated with specific Topoisomerase inhibitors, the TIMP-1 high expressing cells exhibited significantly more resistance to all three inhibitors compared to TIMP-1 low expressing cells. When cells were treated with a combination of SN-38 and epirubicin, we observed an additive but not a synergistic effect. At last, cells were treated with cisplatin with no different effect on TIMP-1 high and low expressing cells. Conclusion and perspectives: The observed upregulation of both protein and phosphorylation levels of Topoisomerases in TIMP-1 high cells may be part of the mechanism by which TIMP-1 confers resistance to treatment with Topoisomerase inhibitors in primary breast cancer. Further work will include pathway analyses and hypothesis testing in clinical material. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-14.

On the origin of prostate fusion oncogenes

A new study reports that androgen signaling induces DNA double-strand breaks and TMPRSS2-ERG rearrangements through androgen receptor–mediated recruitment of topoisomerase 2B. These findings shed light on the generation of the most common fusion oncogene in human cancer.

Effect of chemotherapy on topoisomerase (TOP) expression and activity in colon carcinoma cells

3749 Background: Aim of this study was the evaluation of TOP expression and activity in human cancer cell lines after administration of anti-tumour drugs. Methods: A lymphocytic cell line (Jurkat) and two colon Adenocarcinoma cells lines (LOVO and HT-29) were treated with 1–10 mcM 5-fluorouracile, oxaliplatin, methotrexate and gemcitabine, either as single agents or in combination. Cells were harvested at baseline and at +2h, +3h, +6h and +24h from the beginning of treatment. After RNA extraction, expression of TOP1, TOP2A, TOP 2B was measured by quantitative real-time RT-PCR and correlated with enzymatic activity. Moreover, the cytotoxic effect of the drugs was evaluated on cell lines by MTT assay. Wherever TOP was evaluated by an enzymatic assay. Results: All tested drugs induced TOP1, TOP2A, TOP2B expression at 2–3 h of incubation, with mRNA values ranging from 107% to 910% of baseline levels. TOP expression returned to baseline values at +6h of incubation. Among the different drugs, gemcitabine, oxali...

Effect of chemotherapy on topoisomerase (TOP) expression and activity in colon carcinoma cells

3749 Background: Aim of this study was the evaluation of TOP expression and activity in human cancer cell lines after administration of anti-tumour drugs. Methods: A lymphocytic cell line (Jurkat) and two colon Adenocarcinoma cells lines (LOVO and HT-29) were treated with 1–10 mcM 5-fluorouracile, oxaliplatin, methotrexate and gemcitabine, either as single agents or in combination. Cells were harvested at baseline and at +2h, +3h, +6h and +24h from the beginning of treatment. After RNA extraction, expression of TOP1, TOP2A, TOP 2B was measured by quantitative real-time RT-PCR and correlated with enzymatic activity. Moreover, the cytotoxic effect of the drugs was evaluated on cell lines by MTT assay. Wherever TOP was evaluated by an enzymatic assay. Results: All tested drugs induced TOP1, TOP2A, TOP2B expression at 2–3 h of incubation, with mRNA values ranging from 107% to 910% of baseline levels. TOP expression returned to baseline values at +6h of incubation. Among the different drugs, gemcitabine, oxaliplatin and the combination of 5-fluorouracile + oxaliplatin or methotrexate were the most effective topoisomerase inductors. The increase of mRNA levels corresponded to an increased topoisomerase activity in vitro. Conclusions: Evaluation of cytotoxic activity in vitro showed the schedule including an inductor of TOP (e.g. gemcitabine) followed after +3 h by TOP1 (topotecan) or TOP 2A (etoposide) inhibitors had a synergistic effect, at variance with other sequential administrations of the 2 drugs. In conclusion, anti-tumor drugs acting on DNA replication or cell metabolism induce TOP1, TOP 2A, TOP2B after 2–3 h. Sequential administration of these anticancer drugs with TOP inhibitors show synergistic activity in vitro. No significant financial relationships to disclose.