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

Efimova EV, Liang H, Pitroda SP, Labay E, Darga TE, Levina V, Lokshin A, Roizman B, Weichselbaum RR, Khodarev NN - Int. J. Radiat. Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Illinois 60637, USA.

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 (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.

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 caspases 8, 9 and 3.

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Clonogenic survival of nu61 (black squares) and SCC61 (white diamonds). Cells were plated at P60 (three dishes per dose) and 18 hours after plating irradiated as indicated in Methods. Data were analysed as described in Methods (see also Results for details). Experiments were repeated three times. Shown are mean values; error bars-standard error (SE).
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fig1: Clonogenic survival of nu61 (black squares) and SCC61 (white diamonds). Cells were plated at P60 (three dishes per dose) and 18 hours after plating irradiated as indicated in Methods. Data were analysed as described in Methods (see also Results for details). Experiments were repeated three times. Shown are mean values; error bars-standard error (SE).

Mentions: 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 Stat1 (Khodarev et al. 2004, 2007). In the current experiments we directly compared in vitro clonogenic survival of SCC61 and nu61. It has been shown previously that the parental SCC61 has very low clonogenic ability (Quiet et al. 1991). We therefore used a relatively low range of doses (between 0 and 5 Gy). As is shown in Figure 1, the major difference between the two cell lines was observed between 0 and 2 Gy as a pronounced shoulder in nu61. We used a biphasic model described in (Hall 1988) and previously used by us for correlation of tumour radioresistance in vitro and in vivo (Weichselbaum and Beckett 1987). We found that between 2 and 5 Gy, D0 values for SCC61 and nu61 were 0.66+/−0.03 and 0.60+/−0.07, respectively (mean ± SE, p > 0.05). Extrapolation number (n) was higher for nu61 compared to SCC61 (3.46 ± 2.36 and 1.43 ± 0.14 respectively; mean ± SE) but these differences were also not significant (p > 0.05). However, estimation of D1 in the dose range between 0 and 2 Gy revealed a significant difference between nu61 and SCC61 (2.75 ± 0.03 and 0.99 ± 0.03; p < 0.05). The larger D1 value in nu61 may be attributed to increased sublethal damage repair (Weichselbaum and Beckett 1987, Wang et al. 2008). 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).


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

Efimova EV, Liang H, Pitroda SP, Labay E, Darga TE, Levina V, Lokshin A, Roizman B, Weichselbaum RR, Khodarev NN - Int. J. Radiat. Biol. (2009)

Clonogenic survival of nu61 (black squares) and SCC61 (white diamonds). Cells were plated at P60 (three dishes per dose) and 18 hours after plating irradiated as indicated in Methods. Data were analysed as described in Methods (see also Results for details). Experiments were repeated three times. Shown are mean values; error bars-standard error (SE).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2690884&req=5

fig1: Clonogenic survival of nu61 (black squares) and SCC61 (white diamonds). Cells were plated at P60 (three dishes per dose) and 18 hours after plating irradiated as indicated in Methods. Data were analysed as described in Methods (see also Results for details). Experiments were repeated three times. Shown are mean values; error bars-standard error (SE).
Mentions: 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 Stat1 (Khodarev et al. 2004, 2007). In the current experiments we directly compared in vitro clonogenic survival of SCC61 and nu61. It has been shown previously that the parental SCC61 has very low clonogenic ability (Quiet et al. 1991). We therefore used a relatively low range of doses (between 0 and 5 Gy). As is shown in Figure 1, the major difference between the two cell lines was observed between 0 and 2 Gy as a pronounced shoulder in nu61. We used a biphasic model described in (Hall 1988) and previously used by us for correlation of tumour radioresistance in vitro and in vivo (Weichselbaum and Beckett 1987). We found that between 2 and 5 Gy, D0 values for SCC61 and nu61 were 0.66+/−0.03 and 0.60+/−0.07, respectively (mean ± SE, p > 0.05). Extrapolation number (n) was higher for nu61 compared to SCC61 (3.46 ± 2.36 and 1.43 ± 0.14 respectively; mean ± SE) but these differences were also not significant (p > 0.05). However, estimation of D1 in the dose range between 0 and 2 Gy revealed a significant difference between nu61 and SCC61 (2.75 ± 0.03 and 0.99 ± 0.03; p < 0.05). The larger D1 value in nu61 may be attributed to increased sublethal damage repair (Weichselbaum and Beckett 1987, Wang et al. 2008). 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Illinois 60637, USA.

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 (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.

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 caspases 8, 9 and 3.

Show MeSH
Related in: MedlinePlus