The natural inhibitor of DNA topoisomerase I, camptothecin, modulates HIF-1α activity by changing miR expression patterns in human cancer cells.

Published OnlineFirst November 19, 2013; DOI: 10.1158/1535-7163.MCT-13-0729

The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1 α Activity by Changing miR Expression Patterns in Human Cancer Cells Davide Bertozzi, Jessica Marinello, Stefano G. Manzo, et al. Mol Cancer Ther 2014;13:239-248. Published OnlineFirst November 19, 2013.

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Molecular Cancer Therapeutics

Cancer Biology and Signal Transduction

The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1a Activity by Changing miR Expression Patterns in Human Cancer Cells Davide Bertozzi1, Jessica Marinello1, Stefano G. Manzo1, Francesca Fornari2, Laura Gramantieri2, and Giovanni Capranico1

Abstract DNA topoisomerase I (Top1) inhibition by camptothecin derivatives can impair the hypoxia-induced cell transcriptional response. In the present work, we determined molecular aspects of the mechanism of camptothecin’s effects on hypoxia-inducible factor-1a (HIF-1a) activity in human cancer cells. In particular, we provide evidence that low concentrations of camptothecin, without interfering with HIF-1a mRNA levels, can reduce HIF-1a protein expression and activity. As luciferase assays demonstrated the involvement of the HIF-1a mRNA 30 untranslated region in camptothecin-induced impairment of HIF-1a protein regulation, we performed microarray analysis to identify camptothecin-induced modification of microRNAs (miRNA) targeting HIF-1a mRNA under hypoxic-mimetic conditions. The selected miRNAs were then further analyzed, demonstrating a role for miR-17-5p and miR-155 in HIF-1a protein expression after camptothecin treatments. The present findings establish miRNAs as key factors in a molecular pathway connecting Top1 inhibition and human HIF-1a protein regulation and activity, widening the biologic and molecular activity of camptothecin derivatives and the perspective for novel clinical interventions. Mol Cancer Ther; 13(1); 239–48. 2013 AACR.

Introduction Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as the main regulator of the cellular response to low oxygen tension. It is a dimer constituted by HIF-1a and HIF-1b subunits, and its cellular level is finely governed by the interplay of enzymatic processes that regulate the ubiquitination and proteasomal degradation of the HIF-1a subunit, primarily mediated by the von Hippel–Lindau protein (1, 2). Under cellular oxygen deprivation, HIF-1a accumulates, translocates into the nucleus, and associates with its b subunits to activate the transcription of several target genes, ultimately leading to hypoxia adaptation and survival response. The association of HIF-1 with pathologic events such as cancer, cardiovascular disorders, and inflammation leads to an increased scientific interest in the development of new therapeutic strategies (3–5). Authors' Affiliations: 1Department of Pharmacy and Biotechnology, University of Bologna; and 2Center for Applied Biomedical Research, S. Orsola-Malpighi University Hospital, Bologna, Italy D. Bertozzi and J. Marinello share first authorship for this article. Corresponding Author: G. Capranico, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 48, 40126 Bologna, Italy. Phone: 39-051-2091209; Fax: 39-051-2091224; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-13-0729 2013 American Association for Cancer Research.

Interestingly, the response to hypoxia can be effectively modulated by three DNA topoisomerase I (Top1) poisons (topotecan, camtothecin-20-ester(S), and 9-glycineamido20(S)-camptothecin HCl) in U251 human glioma cells (6); however, the molecular mechanism has not been identified yet. Top1 is a nuclear enzyme that can relax negative and positive DNA supercoils (7), and is the specific target of the natural product camptothecin and its synthetic derivatives, which are effective antitumor drugs (8–10). The pharmacologic activity of camptothecin analogs is due to the drug’s ability to stabilize Top1-DNA cleavage complexes (Top1cc) at replication forks thus leading to collision with DNA polymerase and irreversible DNA damage and apoptosis (11–13). Besides the main interference with the replication process, in recent years it has been studied as the cellular response to Top1ccs formation at transcribed genomic regions (9, 14, 15), as the bulk of cellular Top1 is localized at transcription sites in mammalian cells (16–19). Nevertheless, camptothecin analogs are also known to be endowed with other biologic activities, including antiangiogenic effects (20), and therefore it has been proposed that modulation of hypoxia response cascade by camptothecin could be relevant for the drug’s antitumor activity (21, 22). The effects of topotecan on the human HIF-1a gene have been previously investigated demonstrating that low doses of drug (500 nmol/L or below) inhibit HIF1a protein accumulation during hypoxia in a time-dependent manner (6, 21). As it has been evidenced that

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topotecan does not affect the level of mRNA transcription, it has been suggested a posttranscriptional level of regulation by the drug (15). The compound does not have any effect on the HIF-1b subunit. In addition to this evidence, we previously demonstrated that camptothecin increases the levels of two antisense transcripts at the 50 and 30 ends of the HIF-1a gene in human cancer cells with a cell type and stress specificity (23–25). These antisense RNAs may be involved in mRNA regulation, providing circumstantial evidence of the interplay between Top1 inhibition and response to hypoxia. Moreover, the transcriptional response to Top1ccs in human cancer cell lines includes hyperphosphorylation and enhanced escape from pausing of RNA Pol II (14, 15, 26), activation of cyclin-dependent kinase (CDK; refs. 14, 23, 27), induction of transcription-dependent DNA double-strand breaks (DSB; refs. 28, 29), chromatin remodeling (9, 23), and increased antisense ncRNAs at divergent CpG promoters (30). Consequently, the inhibition of Top1 activity can lead to several alterations of transcription regulation that directly or indirectly may impact on the hypoxia pathways. In this work, we aimed at the definition of molecular factors connecting Top1ccs with HIF-1a protein regulation and activity. In particular, we focused on posttranscriptional molecular mechanisms involving microRNAs (miRNAs) and the 30 untranslated region (30 UTR) of the HIF-1a mRNA. As some camptothecin derivatives have been approved for cancer therapy, the additional evidence that these compounds are able to overcome the effects of HIF-1a accumulation in hypoxic cells may lead to a better understanding of the antiangiogenic and antitumor activity of camptothecin analogs. The new data may lead to different approaches to develop novel therapeutics for the treatment of human cancer and other diseases.

Materials and Methods Cell lines and treatments The cancer cell lines HeLa and HEK293 were purchased from American Type Culture Collection (LGC Standards S.r.l.) 4 years before this publication and were grown in Dulbecco’s Modified Eagle Medium (DMEM; HeLa) or Minimum Essential Medium (MEM; HEK293) with 10% FBS (M-Medical S.r.l.). Cells were maintained at 37 C in a humidified incubator containing 20% O2 and 5% CO2. Cell line identity was periodically certified with the Cell ID System (Promega) by BMR Genomics S.r.l.. Exponentially growing cells were exposed to 0.5 mmol/L of camptothecin for the indicated times at 37 C, unless specified otherwise. In case of cotreatments, cells were incubated with desferrioxamine (250 mmol/L) in the presence of camptothecin (0.5 mmol/L) for the indicated time. Drugs were purchased from Sigma. RNA extraction and reverse transcription After treatment, cells were washed twice with icecold PBS and collected through centrifugation. The

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pellet was resuspended in 3.6 mL of Acetate-EDTA Buffer [50 mmol/L NaOAc (pH 5.2), 10 mmol/L EDTA] containing 240 mL of SDS 25% and 3.6 mL of acid phenol (pH 4.5) and mixed vigorously every minute for 10 minutes at 65 C. After a short incubation on ice, samples were centrifuged for 15 minutes at 12,000 g. The upper phase was collected and added to 3.9 mL of chloroform/isoamyl alcohol then mixed and centrifuged for 10 minutes at 5,000 g. The resulting upper phase was then isopropanol precipitated. DNA was digested with DNase I, and RNA was phenol-extracted and ethanol precipitated. After verifying its quality on a 1% agarose gel, 1 mg of total RNA was used to prepare cDNA using SuperScript III (Life Technologies) following the manufacturer’s instruction. Random (N6) and poly(T) primers were used for total RNA retrotranscription. Reactions included a 25 C preannealing step for 5 minutes, and then retrotranscription was performed at 50 C for 50 minutes. Quantitative real-time PCR Real-time PCR (RT-PCR) was performed using the LightCycler and the FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Quantification and melting curve analyses were performed using the Roche LightCycler software as indicated by the supplier. PCR reactions contained 1 FastStart DNA SYBR Green I Master Mix, 2.08 mmol/L MgCl2 and 350 nmol/L of each primer. Specificity of PCR products was routinely controlled by melting analysis and agarose gel electrophoresis. Differently from the above-specified protocol, miR expression was determined retrotranscribing with the TaqMan MicroRNA RT Kit (Applied Biosystem) and quantifying by the StepOne Real-Time PCR System (Applied Biosystem) with the TaqMan microRNA Assays Kit (Applied Biosystem). In particular, every reaction of 20 mL contains 1 TaqMan Universal Master PCR Mix, 1 TaqMan MicroRNA Assay Mix, and 1.33 mL of cDNA. For primers sequences, see Bertozzi and colleagues (24). Western blot analyses Cells were washed with PBS and lysed for 15 minutes at 4 C with 50% radioimmunoprecipitation assay (RIPA) buffer (Tris-HCl pH 7.4 50 mmol/L, NaCl 150 mmol/L, EDTA 1 mmol/L, NaF 1 mmol/L, sodium deoxycholate 1%, Triton X-100 1%, SDS 0.1%) and 50% HNTG Buffer (HEPES pH 7.4 50 mmol/L, NaCl 150 mmol/L, Triton X-100 0.1%, glycerol 10%) in the presence of protease inhibitors. The lysate was transferred in a tube and centrifuged for 20 minutes, maximum speed. The supernatant was recovered and quantified by Bradford Protein Assay. Western blotting analyses were conducted using appropriate antibodies and the levels of cellular proteins were visualized with peroxidasecoupled secondary antibodies using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific). b-Actin antibody was from Santa Cruz Biotechnology and




Camptothecin Interferes with HIF-1a via microRNAs

HIF-1b antibody was from Novus Biologicals. For HIF-1a detection, BD Biosciences antibody was used. Cells transfection and luciferase assay Functional assay for 30 UTR-HIF-1a. Twenty-four hours after seeding (80,000 cells in each well of a 24-wells plate), cells were transfected with plasmid pRL (renilla luciferase control reporter, 0.5 mg) and pGL3-30 UTRHIF-1a or pGL3-stiI (0.5 mg), using Lipofectamine 2000 (Life Technologies) following the manufacturer’s instructions. Five hours after transfection, the medium was replaced with fresh one and, after additional 19 hours, the drug treatment was started. Finally, cells were lysed with 100 mL of lysis buffer 1 (Promega) for 15 minutes under agitation. Functional assay for HIF-1a activity. Twenty-four hours after seeding (60,000 cells in each well of a 24wells plate), cells were transfected with pRL (0.04 mmol/L), pGL3-HRE-Luc (0.2 mmol/L) and, if indicated, 55 pmol of specific anti-miR (TaqMan anti-miR inhibitor; Life Technologies). Transfection was performed with RNAiMAX (Life Technologies) following manufacturer’s instructions. Twenty-four hours after transfection, medium was replaced with fresh medium completed with drugs. Finally, cells were lysed with 100 mL of lysis buffer 1 (Promega) for 15 minutes under agitation. For the luminometer reading, 100 mL of Luciferase Assay Reagent II (Promega) were added to 20 mL of cellular lysate. After the first reading, 100 mL of Stop & Glo Reagent (Promega) were added and renilla luminescence was determined. miRNA microarray and data analysis RNAs were hybridized on a Agilent Human miRNA microarray (#G4470B; Agilent Technologies). This microarray consists of 60-mer DNA probes synthesized in situ and contains 15,000 features that represent 723 human miRNAs, sourced from the Sanger miRBASE public database (Release 10.1). One-color miRNA expression was performed according to the manufacturer’s procedure, as described in Ferracin and colleagues (31). Briefly, total RNA fraction was obtained from samples by using the acid phenol method as previously described in this section. RNA quality was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies). Low-quality RNAs (RNA integrity number below 7) were excluded from microarray analyses. Labeled miRNAs were obtained from 500 ng of total RNA through the ligation of a 50 -cytidine bisphosphateCy3 (pCp-Cy3; Agilent Technologies) group at the 30 end of each miRNA. To enhance the T4 RNA-ligase (Promega) efficiency, total RNA was previously treated with alkaline phosphatase (Amersham) at 37 C for 30 minutes. Labeled miRNAs were purified by chromatography columns (Micro Biospin 6; Biorad) and then hybridized on a microarray. Hybridizations were performed at 55 C for 17 hours in a rotating oven. Images at 5-mm resolution were generated by the Agilent scanner and


the Feature Extraction 10.5 software (Agilent Technologies) was used to obtain the microarray raw data. Raw data from one-color miRNA microarrays were normalized and analyzed by the GeneSpring GX software version 11.5.1 (Agilent Technologies). The value of each miRNA was obtained doing the average of replicates. Differentially expressed miRNAs were identified by using a filter based on a fold change of 1.45 combined with an ANOVA (P < 0.05) with Benjamini and Hochberg correction for false-positive reduction. Raw and normalized data have been submitted to ArrayExpress with the accession number E-MTAB-2022.

Results Camptothecin inhibition of HIF-1a accumulation under hypoxic-mimetic conditions depends on the 30 UTR of HIF-1a mRNA To elucidate the mechanism of the camptothecininduced reduction of HIF-1a protein levels and activity, we first performed time course experiments of HIF-1a protein accumulation triggered by desferrioxamine, an iron chelator that mimics the response to hypoxia, in HeLa and HEK293 cells. Western Blot analyses of cell lysates demonstrated that a low concentration of camptothecin (0.5 mmol/L) strongly prevents the desferrioxamine-dependent HIF-1a accumulation after 8 to 24 hours (Fig. 1A), whereas it does not affect HIF-1b levels (Fig. 1A, left). The results are thus in agreement with the specific effect of camptothecin on the a subunit of the HIF-1 factor (6, 21). We then measured camptothecin effects on HIF-1a activity with a reporter assay by using a vector expressing a luciferase gene under the control of hypoxia responsive elements (HRE). Cells were transfected with the vector, and after 24 hours treated with desferrioxamine and camptothecin for additional 24 hours. Desferrioxamine treatment induces a strong increase in luciferase activity that is reversed by 0.5 mmol/L camptothecin up to 50% and 20% in HEK293 and HeLa cells, respectively (Fig. 1B). To assess the activity of HIF-1a, we also determined by quantitative RT-PCR (qRT-PCR) the transcriptional activation of the endogenous VEGF gene following cell exposure to desferrioxamine. Camptothecin reduces desferrioxamineactivated VEGF expression in both cell lines after 6 and 24 hours of treatment (Fig. 1C), whereas in normoxic condition camptothecin does not affect the VEGF mRNA level. In contrast, levels of HIF-1a mRNA in HeLa cells are not reduced significantly under hypoxic-mimetic conditions in the presence of camptothecin (Fig. 1D). HEK293 cells show instead a different kinetics, with a decrease at short times of treatment and an increase at longer times. The latter effect could be due to a homeostatic cellular response to the camptothecin-induced protein reduction. Thus, the camptothecin-dependent decrease of HIF-1a protein activity, confirmed by VEGF mRNA reduction in both cell lines,



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A

HeLa

120 kDa

HIF-1α

86 kDa

HIF-1β

HEK293

120 kDa

HIF-1α

42 kDa

β-Actin CPT DFX

B

HEK293

Relative luciferase activity (%)

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CPT DFX

C Relative RNA levels (VEGF)

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D Relative RNA levels (HIF-1α)

2.5

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2

1.5

1.5

1

1

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CPT

Figure 1. Camptothecin (CPT) activity under hypoxic-mimetic conditions in human cancer cells. A, HIF-1a content was determined in HeLa and HEK 293 cells by Western blotting in the presence or absence of desferrioxamine (DFX) and camptothecin (250 and 0.5 mmol/L, respectively). HIF-1b and b-actin are loading controls. B, HIF-1a activity was determined in HeLa and HEK 293 cells transfected with a plasmid expressing the luciferase gene under the control of HREs. Twentyfour hours after transfection, cells were treated either with desferrioxamine alone or desferrioxamine and camptothecin (see above concentrations) for 24 hours. Values are normalized against a control renilla-expressing vector, and over desferrioxaminetreated samples. Values, means SD of four determinations from at least three independent experiments. Levels of VEGF (C) and HIF-1a (D) mRNAs in HeLa and HEK 293 cells following 6 and 24 hours of 0.5 mmol/L camptothecin treatments (independent experiments). Values are normalized to cytochrome B mRNA levels. Camptothecin-treated samples are normalized to untreated samples, whereas camptothecin þ desferrioxamine cotreated samples are normalized to desferrioxamine-treated samples. Values, means SD of four determinations from two independent experiments. , data that are statistically significant (P < 0.05; t test). Lack of genomic DNA contamination was confirmed by a-sat DNA quantitation in each experiment.

DFX 6h

24 h

cannot be attributed to a marked reduction of HIF-1a mRNA levels in HeLa and in HEK293 cell lines. To further investigate if this modulation could be in some way influenced by the previously demonstrated camptothecin stimulation of 30 or 50 -antisense transcripts (30 and 50 aHIF-1a) at the human HIF-1a gene locus (23, 24), we evaluated the level of the two antisense RNAs in the absence and presence of desferrioxamine in both cell lines (Fig. 2). The results demonstrated that no significant increase of the 50 aHIF-1a level is detected after 6 and 24 hours of treatment with a low camptothecin concentration under hypox-

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ic-mimetic conditions (Fig. 2A). A similar situation has been evidenced for 30 aHIF-1a in HeLa cells (Fig. 2B), whereas the transcript level in HEK293 cells is even significantly reduced in hypoxia. Therefore, camptothecin modulation of HIF-1a protein levels seems unlikely to be under the control of the two long noncoding antisense RNAs at the HIF-1a gene locus. As the above results suggested that camptothecin inhibition of HIF-1a accumulation and activity may likely be due to posttranscriptional events, we then assessed whether the 30 UTR of HIF-1a mRNA is involved in the mechanism. We transfected cells with





A Relative RNA levels (5′ aHIF-1α)

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1

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0.5

0

0

a construct in which the HIF-1a 30 UTR was linked to a luciferase gene under the control of the early SV40 promoter, and then cells were treated for 16 hours with desferrioxamine and/or camptothecin. Interestingly, in hypoxic-mimetic conditions, camptothecin treatment markedly reduces luciferase activity in both cell lines (Fig. 3), suggesting a 30 UTR-dependent mechanism for the camptothecin effect. In addition, camptothecin significantly reduces luciferase activity in normoxia in HEK293 but not in Hela cells, indicating that the mechanism is also operative under normoxia at least in


CPT DFX

3

6h

150

HEK293

3

2.5

B Relative RNA levels (3′ aHIF-1α)

Figure 2. Transcription levels of 50 (A) or 30 -antisense RNAs (B) at the HIF-1a gene locus in HeLa and HEK293 cells treated with desferrioxamine and/or camptothecin for the indicated times. Values are normalized to cytochrome B mRNA levels. Camptothecin-treated samples are normalized to untreated samples, whereas camptothecin þ desferrioxamine cotreated samples are normalized to desferrioxamine-treated samples. Values, means SD of four determinations from two independent experiments. , data that are statistically significant (P < 0.05; t test). Lack of genomic DNA contamination was confirmed by a-sat DNA quantitation in each experiment.

HeLa

3

HeLa

24 h

HEK293 cells (Fig. 3). The results overall show that the 30 UTR plays an important role in the reduction of the luciferase activity and likely of HIF-1a protein regulation under hypoxic-mimetic conditions. Low camptothecin concentrations modulate global patterns of miR expression in human HeLa cells As camptothecin-stabilized Top1ccs have been demonstrated to widely interfere with transcriptional mechanisms at active promoters (23, 30), it is likely that this interference may modulate gene expression and

HEK293

150

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100

50

50

0

0 Control

CPT DFX

DFX CPT

Figure 3. HIF-1a 30 UTR is involved in camptothecin-mediated decrease of HIF-1a protein in hypoxic-mimetic conditions. HeLa and HEK 293 cell lines have been transfected with a luciferase plasmid carrying the 30 UTR of HIF-1a. Twenty-four hours after transfection, cells were treated either with desferrioxamine alone (250 mmol/L) or desferrioxamine and camptothecin (250 and 0.5 mmol/L respectively) for 16 hours. Values are normalized to a control renilla-expressing vector and untreated samples. Values, means SD of four determinations from at least three independent experiments. , data that are statistically significant (P < 0.05; t test).




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Table 1. miRNA modulation by camptothecin in normoxic and hypoxic-mimetic condition (fold change >1.45). Normoxia

Hypoxia

Upregulated

Downregulated

Upregulated

Downregulated

68 (5) 5a

6 (2) —

33 (5) 2b

12 (1) 1c

Total Targeting HIF-1a

NOTE: Together with the total number of regulated miRNAs, in parentheses are reported the number of miRNAs emerged as differentially regulated by camptothecin after the ANOVA test (P 0.05) with Benjamini and Hochberg correction. a hsa-miR-15b ; hsa-miR-376c; hsa-miR-18a; hsa-miR-155; hsa-miR-18b. b hsa-miR-376c; hsa-miR-155. c hsa-miR-582-5p.

determine several cascade events. The above evidence that HIF-1a 30 UTR mediates the camptothecin-induced reduction of luciferase (Fig. 3) suggested an involvement of miRNAs in downregulation of mRNA translation upon camptothecin treatments of cells. To study whether Top1ccs may affect miR expression, we conducted a genome-wide analysis of miR expression patterns with the Agilent miRNA microarray platform in HeLa cells treated with a low dose of camptothecin for 6 hours. The drug effect was evaluated both in normoxic and hypoxic-mimetic conditions. To identify the miRNAs that are differentially expressed between camptothecintreated and control cells, we did a statistical comparison between the two groups of samples using a filter based on a fold change of 1.45 (for the complete list of miRNAs emerged by the analysis refer to Supplementary Table S1). Sixty-eight and six miRNAs emerged as up- and downregulated, respectively, by camptothecin in normoxia (Table 1). Similar analysis was performed for desferrioxamine and camptothecin cotreated samples, and in this case 33 and 12 miRNAs resulted up- and downregulated, respectively (Table 1). To highlight the differentially expressed miRNAs in both oxygen conditions, we analyzed the two categories and we revealed that 22 and 2 of the upregulated and downregulated transcripts, respectively, are in common between the normoxic and hypoxic-mimetic conditions (Supplementary Fig. S1). An ANOVA test (P 0.05) with Benjamini and Hochberg correction for false-positive reduction was performed on the miRNAs emerged as differentially regulated by camptothecin. The ANOVA test left fewer miRNAs significantly altered in each category (Table 1, values between bracket). Thus, the analyses show that camptothecin is able to modify specifically the miRNA pool in HeLa cells, with a tendency to upregulate their expression levels. Then, we predicted all the HIF-1a–targeting miRNAs by using miRanda (http://www.microrna.org/microrna/ home.do), TargetScan (http://www.targetscan.org/), PicTar (http://pictar.mdc-berlin.de/), and Diana (http:// diana.cslab.ece.ntua.gr/) algorithms. We next intersected

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the microarray experimental data (not corrected for false-positive reduction, as the ANOVA test resulted in an extremely shorter list) with the in silico predicted HIF-1a–targeted miRNAs, revealing that five and two camptothecin upregulated miRNAs target HIF-1a in normoxia and hypoxic-mimetic conditions, respectively (Table 1). The most significantly upregulated miR was miR-155, which was therefore selected for further analyses. In addition, microarray data suggested that miR-155 could not be the only player in the modulation of HIF-1a levels as several other HIF-1a–targeted miRNAs showed interesting behavior. In fact, two miRNAs derived from the same cluster (miR-18a and miR-18b) were upregulated by camptothecin (Table 1). We therefore selected miR-18a for further validation. Finally, we decided to select one miRNA (miR-17-5p), which is upregulated by camptothecin even if the level of upregulation was not above the 1.45 fold-change threshold, as miR-17-5p targets HIF-1a 30 UTR at 1.1 kb upstream to the 30 end of mRNA, and has a basal expression level higher that the other selected miRNAs. miRNAs are involved in the camptothecin-induced HIF-1a protein reduction in desferrioxamine-treated cells Next, we have investigated the selected miR-155, miR-18a, and miR-17-5p by qRT-PCR in the two human cell lines. Camptothecin can readily increase the levels of miR-155, miR-17-5p, and miR-18a in HeLa cells in both hypoxic-mimetic and normoxic conditions (Fig. 4A). In the presence of desferrioxamine, the effects of camptothecin are dependent on the treatment time, with a stronger increase at 24 hours than 6 hours. Similar analyses in HEK293 cells demonstrated some differences in the kinetics of the miRNAs increase. miR-155 transcription is stimulated by camptothecin in normoxic condition and not significantly in the presence of desferrioxamine. On the contrary, miR-17-5p slightly increases after 24 hours but not after 6 hours of camptothecin treatment under both conditions. miR-18a shows a pattern similar to miR-155, with a stimulation





A

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miR-17-5p

4 3

3

2

2

2

1

1

1

B

– –

+ –

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0

– –

+ –

0

+ +

– –

+ –

– +

+ CPT + DFX

HEK293 miR-155

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miR-18a

4

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1

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+ –

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+ +

0

– –

+ –

6h

in normoxia and no effects in the presence of desferrioxamine (Fig. 4B). The effect of desferrioxamine on the expression of the studied miRNAs has also been evaluated highlighting the fact that transcription of miR-18a is induced by 6 hours of hypoxia in both cell lines but not at longer times (Supplementary Fig. S2), whereas miR-17-5p is not modified or partially reduced in HeLa and HEK293 cells, respectively (Supplementary Fig. S2). Differently, the miR-155 transcript level increases after 24 hours of desferrioxamine treatment in HeLa cells. The results are therefore in agreement with the microarray data in HeLa cells, suggesting that camptothecin may modulate the HIF-1a protein level through the action of different miRNAs. If the selected and analyzed miRNAs are involved in the observed camptothecin-dependent reduction of HIF-1a, then specific anti-miR expression should suppress this effect. Thus, we next performed experiments in which cells were transfected with a plasmid with the luciferase gene under the control of HRE, together with an anti-miR against one of the selected miR-155, miR-18a, or miR17-5p. Then, 24 hours after transfection, cells were treated either with desferrioxamine alone or with desferrioxamine and camptothecin for 24 hours. As previously reported in Fig. 1B, first of all, we can observe that the treatment of desferrioxamine alone significantly increases the luciferase activity compared with control cells, confirming the activation of hypoxia response pathways (Fig. 5). After cotreatment with desferrioxamine and camptothecin, we observe a reduction of the luciferase activity in both cell lines (black


– +

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4

3

0

Relative miR expression

Figure 4. Expression levels of selected miRNAs in HeLa (A) and HEK 293 (B) cell lines in normoxic and hypoxic-mimetic conditions after 6 and 24 hours of camptothecin treatments. Values are normalized to U6RNA levels. Camptothecin-treated samples are also normalized to untreated samples, whereas camptothecin þ desferrioxamine–treated samples are normalized to desferrioxaminetreated samples. Values, means SD of four determinations from two independent experiments. , data that are statistically significant (P < 0.05; t test).

Relative miR expression

4

– +

0

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– –

+ –

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24 h

bars, no anti-miR, Fig. 5). Then, we determined the effect of the selected miRs by transfecting the corresponding anti-miR. The results show that anti-miR17-5p is able to suppress the camptothecin-reduced luciferase activity in both cell lines, whereas anti-miR-155 is effective to a lesser extent (Fig. 5). In contrast, the anti-miR-18a was ineffective, whereas the combination of all the tested anti-miRs gave a full suppression of camptothecin effects (Fig. 5). Taken together, the present results show that, in hypoxic-mimetic conditions, camptothecin can inhibit HIF-1a protein accumulation by the modulation of miRNAs targeting the HIF-1a mRNA. Here, we have shown that miR-17-5p and miR-155 can be among those HIF-1a–targeted miRNAs altered by low doses of camptothecin.

Discussion The present report defines the molecular players connecting Top1ccs with HIF-1a protein regulation and activity, demonstrating that Top1 inhibitors can be sharp regulators of gene activity. Camptothecin and its derivatives have been deeply studied since their discovery, and for several decades their cytotoxic activity was strictly associated only with irreversible replicative DNA damage leading to S-phase checkpoint activation, G2 arrest, and cell death. In the past few years, the attention has been oriented back on these chemical inhibitors as several additional effects have been highlighted, mostly reflected on the interference with transcription. We evidenced that Top1 inhibition favors



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160 140 120 100 80 60 40 20 0

No anti-miR

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+ + Anti-17-5p

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– – Anti-18a

– + Anti-155

+ +

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Anti-miR all

Figure 5. Anti-miR-17-5p reverts camptothecin-induced HIF-1a activity reduction in hypoxic-mimetic conditions both in HeLa and HEK 293 cell lines. Cells have been cotransfected with a luciferase plasmid under the control of HREs and with the indicated anti-miR. Twenty-four hours after transfection, cells were treated either with desferrioxamine alone (250 mmol/L) or desferrioxamine and camptothecin (250 and 0.5 mmol/L, respectively) for further 24 hours. Values are normalized to control renilla-expressing vector and desferrioxamine-treated samples. Values, means SD of four determinations from at least three independent experiments. , data that are statistically significant (P < 0.05; t test).

RNA Pol II escape from the promoter-proximal pausing site of several genes, enhances histone acetylation and reduces nucleosome density in a CDK-dependent manner, and finally induces alterations of splicing events (9, 14, 23, 28, 29, 32). It is noteworthy that camptothecin can increase genome-wide the level of antisense RNAs at CpG island promoters of divergent genes (30) and, specifically for the present work, it is able to induce the transcription of long noncoding antisense RNAs at the 50 and 30 end of the HIF-1a gene (23, 24), which may regulate gene expression posttranscriptionally (24). Camptothecin can also activate an ataxia telangiectasia mutated (ATM)- and DNAdependent protein kinase (DNA-PK)-dependent transcriptional response in quiescent cells, such as neurons (28, 29), likely leading to DNA breaks and transcriptionassociated DNA rearrangements (33). Finally, Top1 inhibitors can unsilence the paternal Ube3a allele in a mouse model, giving the possibility to restore functional UBE3A protein and treat Angelman syndrome (34, 35). Thus, several findings on camptothecin activity not related to replicative DNA damage and cell killing of S-phase cells prompted us to define transcriptional molecular response triggered by Top1ccs and affecting HIF-1a gene activity. Here, we have shown the role of selected miRNAs in the molecular interference of low camptothecin concentrations with HIF-1a protein accumulation under hypoxic-mimetic conditions. The drug treatment strongly prevents the protein accumulation as detected by western blotting and luciferase assay, even though the two methods show different sensitivity. The camptothecin-induced impairment of HIF-1a accumulation impacts the hypoxia-mediated cascade of events, with a decrease of HIF-1a activity and VEGF mRNA transcription. Interestingly, the studied low concentrations of camptothecin do not interfere with HIF-1a transcript levels, in agreement with a posttranscriptional miR-

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based mechanism of HIF-1a reduction. Interestingly, camptothecin can significantly alter miR expression profiles, and in particular can increase miR-17-5p and miR-155, therefore affecting HIF-1a activity in human cancer cells. In recent years, many miRNAs targeting complementary sequences in 30 UTR have been discovered, and evidence has much increased showing their importance in regulating physiologic and pathologic cellular pathways (36). As camptothecin treatment determines strong modification of transcription of several mRNAs (30, 37, 38), we questioned if the drug could modulate the miRNAs expression profiles in two human cancer cell lines and, if that was the case, if miRNAs modifications reflect in the HIF-1a protein expression. Microarray results pushed us to further investigate the role of three selected miRNAs in the modulation of HIF-1a protein accumulation in the presence of camptothecin. qRT-PCR confirmed an increased levels of the miRs under the experimental conditions. Interestingly, luciferase experiments performed after antimiR transfections demonstrated that miR-17-5p and miR-155 can be two important players in the reduction of HIF-1a protein accumulation and activity by low camptothecin concentrations. Western blot experiments showed modest alterations of camptothecin effects in the presence of anti-miRs (not shown). This could be due to different sensitivity between methodologies in assessing HIF-1a protein levels, but also to the involvement of different posttranscriptional mechanisms of HIF-1a regulation. For example, it has been demonstrated that HuR and PTB RNA-binding proteins can also cooperate and enhance HIF-1a translation by binding to the 50 UTR of HIF-1a mRNA (39); therefore, they may overcome the effect of the studied anti-miRs in the case of HIF-1a mRNA. In addition, the results overall indicate that other miRNAs, besides the ones studied in the present work, can likely be involved in the





mechanism of HIF-1a interference by camptothecin with a cell-type specificity. Here, we have demonstrated for the first time that Top1ccs can modulate HIF-1a via miRNAs in human cancer cell lines. Recent data (40) demonstrated that higher concentrations of camptothecin affect few miRNAs in normoxic condition in HCT116 cells, and between them, miR-142-3p was the most significantly upregulated with a consequent downregulation of hundreds of predicted target genes, as those of "ubiquitin-mediated proteolysis" gene family. Interestingly, modifications of levels of several miRNAs after camptothecin treatments in normoxic cancer cells have been related to resistance or sensitivity to Top1 inhibitors (41–44). Overall, these studies confirm that different concentrations of Top1 inhibitors finely modulate the balance of cellular miRNAs levels. In addition to mentioned data, the present report shows that Top1 inhibition impairs the normal level of miRNAs not only in normal but also in hypoxic-mimetic conditions. The reported miRNA expression profile alterations could impact different cellular pathways besides the cellular response to hypoxia. In conclusion, the present study aimed at clarifying the molecular factors connecting the Top1 inhibition with HIF-1a protein regulation and activity. The findings demonstrate that camptothecin cellular effects are associated not only with replicative DNA damage, G2 arrest, and programmed cell death, but also with the transcription-specific miR-based response. Our findings, showing that low camptothecin concentrations can finely modulate miR expression profiles, indicate that camptothecin anticancer and antiangiogenic activ-

ity can be due to a more specific transcription response involving small noncoding RNA, such as miR-17-5p and miR-155. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Authors' Contributions Conception and design: D. Bertozzi, J. Marinello, S.G. Manzo, G. Capranico Development of methodology: D. Bertozzi, G. Capranico Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Bertozzi, J. Marinello, S.G. Manzo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Bertozzi, J. Marinello, S.G. Manzo, F. Fornari, L. Gramantieri, G. Capranico Writing, review, and/or revision of the manuscript: D. Bertozzi, J. Marinello, G. Capranico Study supervision: D. Bertozzi, G. Capranico

Acknowledgments The authors thank the Centro Interdipartimentale per le Ricerche Biotecnologie (CIRB) of Bologna University for equipment facilities. They also thank M. Ferracin and the Ferrara Functional Genomics (Microarray Facility at Dipartimento Medicina Sperimentale e Diagnostica—University of Ferrara).

Grant Support This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy (grant IG10184; to G. Capranico), and the University of Bologna PhD Program in Cellular and Molecular Biology (to D. Bertozzi and S.G. Manzo). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received August 30, 2013; revised October 28, 2013; accepted November 11, 2013; published OnlineFirst November 19, 2013.

References 1.

2.

3. 4.

5. 6.

7. 8. 9.

Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 1996;271:17771–8. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:5510–4. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721–32. Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, et al. Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases. Cancer Res 1999;59:5830–5. Onnis B, Rapisarda A, Melillo G. Development of HIF-1 inhibitors for cancer therapy. J Cell Mol Med 2009;13:2780–6. Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 2002;62:4316–24. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 2001;70:369–413. Pommier Y. Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 2006;6:789–802. Capranico G, Marinello J, Baranello L. Dissecting the transcriptional functions of human DNA topoisomerase I by selective inhibitors: implications for physiological and therapeutic modulation of enzyme activity. Biochim Biophys Acta 2010;1806:240–50.


10. Pommier Y. Drugging topoisomerases: lessons and challenges. ACS Chem Biol 2012;8:82–95. 11. Pommier Y, Barcelo JM, Rao VA, Sordet O, Jobson AG, Thibaut L, et al. Repair of topoisomerase I-mediated DNA damage. Prog Nucleic Acid Res Mol Biol 2006;81:179–229. 12. Staker BL, Hjerrild K, Feese MD, Behnke CA, Burgin ABJr., Stewart L. The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci U S A 2002;99:15387–92. 13. Ricci A, Marinello J, Bortolus M, Sanchez A, Grandas A, Pedroso E, et al. Electron paramagnetic resonance (EPR) study of spin-labeled camptothecin derivatives: a different look of the ternary complex. J Med Chem 2011;54:1003–9. 14. Khobta A, Ferri F, Lotito L, Montecucco A, Rossi R, Capranico G. Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells. J Mol Biol 2006;357:127–38. 15. Lavelle C. Transcription elongation through a chromatin template. Biochimie 2007;89:516–27. 16. Kretzschmar M, Meisterernst M, Roeder RG. Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc Natl Acad Sci U S A 1993; 90:11508–12. 17. Tuduri S, Crabbe L, Conti C, Tourriere H, Holtgreve-Grez H, Jauch A, et al. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat Cell Biol 2009;11:1315–24.



247


Bertozzi et al.

18. Wu HY, Shyy SH, Wang JC, Liu LF. Transcription generates positively and negatively supercoiled domains in the template. Cell 1988;53:433–40. 19. Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 2002;3:430–40. 20. Guerin E, Raffelsberger W, Pencreach E, Maier A, Neuville A, Schneider A, et al. In vivo topoisomerase I inhibition attenuates the expression of hypoxia-inducible factor 1 alpha target genes and decreases tumor angiogenesis. Mol Med 2011;18:83–94. 21. Rapisarda A, Uranchimeg B, Sordet O, Pommier Y, Shoemaker RH, Melillo G. Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism and therapeutic implications. Cancer Res 2004; 64:1475–82. 22. Rapisarda A, Zalek J, Hollingshead M, Braunschweig T, Uranchimeg B, Bonomi CA, et al. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res 2004;64:6845–8. 23. Baranello L, Bertozzi D, Fogli MV, Pommier Y, Capranico G. DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1alpha gene locus. Nucleic Acids Res 2009;38:159–71. 24. Bertozzi D, Iurlaro R, Sordet O, Marinello J, Zaffaroni N, Capranico G. Characterization of novel antisense HIF-1alpha transcripts in human cancers. Cell Cycle 2011;10:3189–97. 25. Thrash-Bingham CA, Tartof KD. aHIF: a natural antisense transcript overexpressed in human renal cancer and during hypoxia. J Natl Cancer Inst 1999;91:143–51. 26. Sordet O, Larochelle S, Nicolas E, Stevens EV, Zhang C, Shokat KM, et al. Hyperphosphorylation of RNA polymerase II in response to topoisomerase I cleavage complexes and its association with transcription- and BRCA1-dependent degradation of topoisomerase I. J Mol Biol 2008;381:540–9. 27. Amente S, Gargano B, Napolitano G, Lania L, Majello B. Camptothecin releases P-TEFb from the inactive 7SK snRNP complex. Cell Cycle 2009;8:1249–55. 28. Sordet O, Nakamura AJ, Redon CE, Pommier Y. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions. Cell Cycle 2009;9:274–8. 29. Sordet O, Redon CE, Guirouilh-Barbat J, Smith S, Solier S, Douarre C, et al. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks. EMBO Rep 2009;10:887–93. 30. Marinello J, Chillemi G, Bueno S, Manzo SG, Capranico G. Antisense transcripts enhanced by camptothecin at divergent CpG-island promoters associated with bursts of topoisomerase I-DNA cleavage complex and R-loop formation. Nucleic Acids Res. Epub 2013 Sep 2.

248


31. Ferracin M, Zagatti B, Rizzotto L, Cavazzini F, Veronese A, Ciccone M, et al. MicroRNAs involvement in fludarabine refractory chronic lymphocytic leukemia. Mol Cancer 2010;9:123. 32. Solier S, Barb J, Zeeberg BR, Varma S, Ryan MC, Kohn KW, et al. Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors. Cancer Res 2010;70:8055–65. 33. Aguilera A, Gomez-Gonzalez B. Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet 2008;9:204–17. 34. Huang HS, Allen JA, Mabb AM, King IF, Miriyala J, Taylor-Blake B, et al. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 2011;481:185–9. 35. Powell WT, Coulson RL, Gonzales ML, Crary FK, Wong SS, Adams S, et al. R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation. Proc Natl Acad Sci U S A 2013;110:13938–43. 36. Yates LA, Norbury CJ, Gilbert RJ. The long and short of microRNA. Cell 2013;153:516–9. 37. Lotito L, Russo A, Bueno S, Chillemi G, Fogli MV, Capranico G. A specific transcriptional response of yeast cells to camptothecin dependent on the Swi4 and Mbp1 factors. Eur J Pharmacol 2009; 603:29–36. 38. Lotito L, Russo A, Chillemi G, Bueno S, Cavalieri D, Capranico G. Global transcription regulation by DNA topoisomerase I in exponentially growing saccharomyces cerevisiae cells: activation of telomere-proximal genes by TOP1 deletion. J Mol Biol 2008;377: 311–22. 39. Galban S, Kuwano Y, Pullmann RJr., Martindale JL, Kim HH, Lal A, et al. RNA-binding proteins HuR and PTB promote the translation of hypoxia-inducible factor 1alpha. Mol Cell Biol 2008;28:93–107. 40. Solier S, Ryan MC, Martin SE, Varma S, Kohn KW, Liu H, et al. Transcription poisoning by topoisomerase I is controlled by gene length, splice sites, and miR-142-3p. Cancer Res 2013;73:4830–9. 41. Bitarte N, Bandres E, Boni V, Zarate R, Rodriguez J, Gonzalez-Huarriz M, et al. MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of colorectal cancer stem cells. Stem Cells 2011;29:1661–71. 42. Gmeiner WH, Reinhold WC, Pommier Y. Genome-wide mRNA and microRNA profiling of the NCI 60 cell-line screen and comparison of FdUMP[10] with fluorouracil, floxuridine, and topoisomerase 1 poisons. Mol Cancer Ther 2010;9:3105–14. 43. Tong JL, Zhang CP, Nie F, Xu XT, Zhu MM, Xiao SD, et al. MicroRNA 506 regulates expression of PPAR alpha in hydroxycamptothecinresistant human colon cancer cells. FEBS Lett 2011;585:3560–8. 44. Zeng CW, Zhang XJ, Lin KY, Ye H, Feng SY, Zhang H, et al. Camptothecin induces apoptosis in cancer cells via microRNA125b-mediated mitochondrial pathways. Mol Pharmacol 2012;81: 578–86.



Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells.

The topoisomerase I inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells.

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

DNA topoisomerase I domain interactions impact enzyme activity and sensitivity to camptothecin.

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

Expression of the type I DNA topoisomerase gene in adenovirus-5 infected human cells.

Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids.

Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Mitochondrial DNA topoisomerase I from human platelets.

Doxorubicin inhibits human DNA topoisomerase I.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.

Activity of topotecan, a new topoisomerase I inhibitor, against human tumor colony-forming units in vitro.

Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer.

Structural characterization of the human DNA topoisomerase I gene promoter.

A DNA topoisomerase I inhibitor blocks the differentiation of rat granulosa cells induced by follicle-stimulating hormone.

Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells.

2α mediates hypoxia-induced LDHA expression in human pancreatic cancer cells.

MiR-489 modulates cisplatin resistance in human ovarian cancer cells by targeting Akt3.

Effect of p53 activity on the sensitivity of human glioblastoma cells to PARP-1 inhibitor in combination with topoisomerase I inhibitor or radiation.