No topoisomerase I alteration in a neuroblastoma model with in vivo acquired resistance to irinotecan

Calvet, Loreley; Santos, Allan de Oliveira; Valent, Alexander; Mj, Terrier-Lacombe; Opolon, Paule; Merlin, J.L.; Aubert, Geneviève; Morizet, Jackie; Schellens, Jan H.M.; Bénard, Jean; Vassal, Gilles

doi:10.1038/sj.bjc.6602079

Cited by 13 publications

(10 citation statements)

References 27 publications

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“…The reduction of Top1 gene expression for drug resistance might be drug structurespecific, because no Top1 alteration was observed in a neuroblastoma model with in vivo-acquired resistance to irinotecan (Calvet et al, 2004). It is reasonable that the two resistant cell lines also show cross-resistance to other Top1 inhibitors because of decreased amount of their drug target, Top1.…”

Section: Discussionmentioning

confidence: 99%

Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan

Liao¹,

Robey²,

Guirouilh‐Barbat³

et al. 2007

Mol Pharmacol

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Homocamptothecins (hCPTs) are a novel class of topoisomerase I (Top1) inhibitors with enhanced chemical stability compared with the currently used camptothecin (CPT) analogs irinotecan and topotecan. The hCPT derivative diflomotecan (BN80915) is currently in clinical trials. We established two resistant human glioblastoma cell lines, SF295/hCPT50 and SF295/BN50, by stepwise exposure of the parental SF295 line to increasing concentrations of hCPT and BN80915, respectively. The two resistant cell lines were 15-to 22-fold resistant to hCPT and BN80915 as well as 7-to 27-fold crossresistant to other Top1 inhibitors, including CPT, topotecan, and the indenoisoquinolines MJ-III-65 (NSC 706744) and NSC 724998, but sensitive to the topoisomerase II inhibitors mitoxantrone and etoposide. Neither of the resistant cell lines displayed any detectable expression of the three major drug transporters P-glycoprotien, multidrug resistance-associated protein 1, or ATP-binding cassette, subfamily G (WHITE), member 2, as assessed by immunoblot or flow cytometry. Reduced expression of Top1 protein occurred at the transcriptional level in both of the resistant cell lines, consistent with the reduction of Top1 enzyme level as the major contribution to the resistance phenotype in SF295/ hCPT50 and SF295/BN50 cells. Treatment of the resistant cell lines with the histone deacetylase inhibitor depsipeptide or the DNA methyltransferase inhibitor 5-aza-2Ј-deoxycytidine alone or concomitantly did not result in re-expression of Top1. Our studies suggest that selection for resistance to hCPT or BN80915 is primarily related to reduced Top1 expression at the transcriptional level, resulting in reduced enzyme levels.Homocamptothecins (hCPTs) are a new class of camptothecin (CPT) derivatives. They are different from the conventional CPT in that they bear a seven-membered ␤-hydroxylactone ring instead of the naturally occurring six-membered ␣-hydroxylactone observed in CPT (see Fig. 1). hCPTs are therefore more stable than CPT derivatives (Lavergne et al., 2000a,b) that are readily hydrolyzed to the inactive carboxylate forms (for review, see Pommier, 2006). hCPT, like other CPT analogs, inhibits Top1 by stabilizing the covalent complex between Top1 and DNA, leading to enzyme-linked DNA strand breaks, also referred to as cleavage complexes (Pommier, 2006). Because of their increased chemical stability, hCPTs were expected to exert greater Top1 inhibition and antitumor efficacy than the current FDA-approved CPT analogs irinotecan and topotecan [for review, see Lavergne et al. (2000b);Pommier (2006)]. hCPTs showed very potent antitumor efficacy in many tumors, including brain glioblastoma mouse models. The fluorinated hCPT diflomotecan (BN80915) is one of the most active compounds in terms of antiproliferative activity in the hCPT series (Bailly et al., 1999;Lavergne et al., 2000b). BN80915 has been selected for further development as a highly active hCPT-based Top1

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Section: Discussionmentioning

confidence: 99%

Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan

Liao¹,

Robey²,

Guirouilh‐Barbat³

et al. 2007

Mol Pharmacol

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“…To date, mechanisms that confer clinical resistance to topoisomerase I inhibitors, such as irinotecan, have not been fully characterized in the clinical setting. Our in vivo NB model resistant to irinotecan is of great interest, because resistance mechanisms usually described in vitro, such as, topoisomerase I alteration and multidrug-resistance-related protein overexpression, were not observed (Calvet et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…In vivo, IGR-NB8 proved to be sensitive to CPT-11 and topotecan. IGR-NB8 was rendered resistant to CPT-11 through prolonged exposure to CPT-11 given at a dose of 27 mg/kg/day Â 5 every 21 days (one cycle) for a maximum of four cycles (one passage) (Calvet et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

“…The acquired resistance of IGR-NB8-R was not permanent. The tumor xenograft recovered its initial sensitivity after 15 passages without treatment (Calvet et al, 2004). For the study, tumor samples from IGR-NB8 (sensitive), IGR-NB8-R (resistant) and IGR-NB8-r (reverted) were rapidly harvested, frozen in nitrogen liquid, and stored at À801C.…”

Section: Methodsmentioning

confidence: 99%

“…Prolonged exposure of a NB xenograft to CPT-11 allowed resistance to be acquired progressively (Calvet et al, 2004). This resistant xenograft (IGR-NB8-R) was devoid of topoisomerase I alteration and of overexpresssion of three members of the ATP-binding cassette (ABC) transporter family that confer multidrug resistance (MDR).…”

Section: Introductionmentioning

confidence: 99%

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Pleiotrophin, a candidate gene for poor tumor vasculature and in vivo neuroblastoma sensitivity to irinotecan

et al. 2006

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In vivo neuroblastoma (NB) xenograft model, resistant to the DNA-topoisomerase I inhibitor irinotecan (CPT-11), has been established to study resistance mechanisms acquired in a therapeutic setting. Common mechanisms of resistance were not involved in this resistance. Thus, we compared the gene expression profiles of sensitive, resistant, and reverted tumors using cDNA expression arrays. Expression of selected transcripts was confirmed by quantitative real-time PCR. We found that pleiotrophin (PTN), a heparin-binding growth factor, was the only gene significantly affected: PTN gene expression was downregulated in all resistant tumors (8-14-fold) as compared to sensitive tumors, and was increased (2-4-fold) in all reverted tumors as compared to resistant tumors. PTN thus appeared to be a likely candidate gene associated with resistance to CPT-11 in this in vivo model. To investigate the direct implication of PTN in NB, we transfected two NB cell lines with RNA interferences in order to silence PTN. PTN failed to demonstrate implication in resistance to CPT-11 in vitro but could influence sensitivity to CPT-11 exclusively through an in vivo mechanism. Indeed, vasculature was significantly enhanced in resistant NB xenografts compared to sensitive and reverted xenografts, and we suggest that PTN is acting in our resistant in vivo NB model as an angiostatic factor.

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Flow cytometry analysis of single‐strand DNA damage in neuroblastoma cell lines using the F7‐26 monoclonal antibody

et al. 2007

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The F7-26 monoclonal antibody (Mab) has been reported to be specific for singlestrand DNA damage (ssDNA) and to also identify cells in apoptosis. We carried out studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC) 6 pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy c radiation 6 hydrogen peroxide (H 2 O 2 ) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM 6 BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-dfmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation 1 H 2 O 2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs. ' 2007 International Society for Analytical Cytology

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No topoisomerase I alteration in a neuroblastoma model with in vivo acquired resistance to irinotecan

Cited by 13 publications

References 27 publications

Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan

Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan

Pleiotrophin, a candidate gene for poor tumor vasculature and in vivo neuroblastoma sensitivity to irinotecan

Flow cytometry analysis of single‐strand DNA damage in neuroblastoma cell lines using the F7‐26 monoclonal antibody

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