Radioresistance of Stat1 over-expressing tumour cells is associated with suppressed apoptotic response to cytotoxic agents and increased IL6-IL8 signalling

Elena V Efimova,Hua Liang,Sean P Pitroda,Edwardine Labay,Thomas E Darga,Vera Levina,Anna Lokshin,Bernard Roizman,Ralph R Weichselbaum,Nikolai N Khodarev

doi:10.1080/09553000902838566

Abstract

Purpose: To determine the mechanisms of Signal Transducer and Activator of Transcription 1 (Stat1)-associated radioresistance developed by nu61 tumour selected in vivo by fractionated irradiation of the parental radiosensitive tumour SCC61.Materials and methods: Radioresistence of nu61 and SCC61 in vitro was measured by clonogenic assay. Apoptotic response of nu61 and SCC61 cells to genotoxic stress was examined using caspase-based apoptotic assays. Co-cultivation of carboxyfluorescein diacetate, succinimidyl ester (CFDE-SE)-labeled nu61 with un-labeled SCC61 was performed at 1:1 ratio. Production of interleukin-6, interleukin-8 and soluble receptor of interleukin 6 (IL6, IL8 and sIL6R) was measured using Enzyme-Linked Immunosorbent Assay (ELISA).Results: Radioresistant nu61 was also resistant to interferon-gamma (IFNγ) and the death ligands of tumour necrosis factor alpha receptor (TNFR) family when compared to SCC61. This combined resistance is due to an impaired apoptotic response in nu61. Relative to SCC61, nu61 produced more IL6, IL8 and sIL6R. Using Stat1 knock-downs we demonstrated that IL6 and IL8 production is Stat1-dependent. Treatment with neutralising antibodies to IL6 and IL8, but not to either cytokine alone sensitised nu61 to genotoxic stress induced apoptosis.Conclusion: Nu61, which over-expresses Stat1 pathway, is deficient in apoptotic response to ionising radiation and cytotoxic ligands. This resistance to apoptosis is associated with Stat1-dependent production of IL6 and IL8 and suppression of 8, 9 and 3.

Highlights

Tumour cells may acquire radioresistance using multiple pathways including those which are induced/altered by ionising radiation (IR) itself (Lee and Bernstein 1993, Tyrsina et al 2005, Otero et al 2006)
In our previous reports we demonstrated that the nu61 tumour, selected from the SCC61 tumour by in vivo fractionated irradiation, is more radioresistant based on in vivo assays and overexpresses Signal Transducer and Activator of Transcription 1 (Stat1) (Khodarev et al 2004, 2007)
Literature indicates that sublethal damage repair is connected with increased resistance to genotoxic stress associated with suppressed apoptosis (Blenn et al 2006, Hara et al 2008)

Summary

Introduction

Tumour cells may acquire radioresistance using multiple pathways including those which are induced/altered by ionising radiation (IR) itself (Lee and Bernstein 1993, Tyrsina et al 2005, Otero et al 2006). To investigate IR-induced tumour radioresistance, a radiosensitive human squamous cell carcinoma tumour, SCC61, was passed and irradiated in vivo. Radioresistant tumours were selected and a tumour cell line, nu, was isolated (Khodarev et al 2004). Analysis of the differences in gene expression between cells from differentially selected tumours demonstrated up-regulation of the genes in the Signal Transducer and Activator of Transcription 1 (Stat1) signalling pathway in radioresistant nu tumours compared with radiosensitive SCC61 tumours. We and others reported that IR up-regulates Stat and a Stat1-dependent pathway in vitro and in vivo (Amundson et al 2004, Khodarev et al 2007) and tumour cells resistant to radiation are resistant to interferon.

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Conclusion