Abstract
Recurrence is frequently associated with the acquisition of radioresistance by tumors and resulting failures in radiotherapy. We report, in this study, that long-term fractionated radiation (FR) exposures conferred radioresistance to the human tumor cells, HepG2 and HeLa with cyclin D1 overexpression. A positive feedback loop was responsible for the cyclin D1 overexpression in which constitutively active AKT was involved. AKT is known to inactivate glycogen synthase kinase-3β (GSK3β), which is essential for the proteasomal degradation of cyclin D1. The resulting cyclin D1 overexpression led to the forced progression of S-phase with the induction of DNA double strand breaks. Cyclin D1-dependent DNA damage activated DNA-dependent protein kinase (DNA-PK), which in turn activated AKT and inactivated GSK3β, thus completing a positive feedback loop of cyclin D1 overproduction. Cyclin D1 overexpression led to the activation of DNA damage response (DDR) consisted of ataxia telangiectasia mutated (ATM)- and Chk1-dependent DNA damage checkpoint and homologous recombination repair (HRR). Long-term FR cells repaired radiation-induced DNA damage faster than non-FR cells. Thus, acquired radioresistance of long-term FR cells was the result of alterations in DDR mediated by cyclin D1 overexpression. Inhibition of the AKT/GSK3β/cyclin D1/Cdk4 pathway by the AKT inhibitor, Cdk4 inhibitor or cyclin D1 targeting small interfering RNA (siRNA) suppressed the radioresistance. Present observations give a mechanistic insight for acquired radioresistance of tumor cells by cyclin D1 overexpression, and provide novel therapeutic targets for recurrent radioresistant tumors.
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Abbreviations
- API-2:
-
AKT/protein kinase B signaling inhibitor-2
- APC:
-
anaphase promoting factor
- BSA:
-
bovine serum albumin
- BrdU:
-
bromodeoxyuridine
- CSCs:
-
cancer stem cells
- CDKs:
-
cyclin-dependent kinases
- CHX:
-
cycloheximide
- DDR:
-
DNA damage response
- DNA-PK:
-
DNA-dependent protein kinase
- DSBs:
-
double strand breaks
- FACS:
-
fluorescence-activated cell sorting
- FR:
-
fractionated radiation
- GSK3β:
-
glycogen synthase kinase-3β
- HE:
-
hematoxylin-eosin
- IHC:
-
immunohistochemistry
- PBS:
-
phosphate-buffered saline
- PI:
-
propidium iodide
- PKB:
-
protein kinase B
- SR:
-
single radiation
- SDS–PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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Acknowledgements
We thank Dr Ohtsura Niwa and Dr Keiko Nakayama for their critical reading of the paper. We thank Dr J Alan Diehl for providing pFleX-cyclin D1 expression vectors. We thank Dr Shuntaro Ikawa for providing some chemicals. This study was in part supported by the Grant-in-Aid for young scientists (Start-up-20810003) and (Wakate-B-21710054), by the Grant-in-Aid from Ministry of Education, Culture, Sports, Science and Technology, by grants from the Ministry of Health, Labour and Welfare of Japan, and by the ‘Frontier Science Research Program’ in the Center for Interdisciplinary Research, Tohoku University.
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Shimura, T., Kakuda, S., Ochiai, Y. et al. Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. Oncogene 29, 4826–4837 (2010). https://doi.org/10.1038/onc.2010.238
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DOI: https://doi.org/10.1038/onc.2010.238
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