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


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 (IFNgamma) 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.


CONCLUSIONS
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 caspases 8, 9 and 3.
Department of Radiation and Cellular Oncology, The University of Chicago, Illinois 60637, USA.
Elena V
EV
Efimova
Hua
H
Liang
Sean P
SP
Pitroda
Edwardine
E
Labay
Thomas E
TE
Darga
Vera
V
Levina
Anna
A
Lokshin
Bernard
B
Roizman
Ralph R
RR
Weichselbaum
Nikolai N
NN
Khodarev
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D018920:Coculture Techniques; D016207:Cytokines / Q000633:toxicity; D003603:Cytotoxins / Q000633:toxicity; D004249:DNA Damage; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects
D016207:Cytokines; D003603:Cytotoxins; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D018124:Receptors, Tumor Necrosis Factor; D050794:STAT1 Transcription Factor; D007371:Interferon-gamma; D020169:Caspases