S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) Theses (Ph.D. / Sc.D._의학과)
Regulation of DNA damage responses by stimulatory G protein signaling in cancer cells
- 의과대학 의학과
- Issue Date
- 서울대학교 대학원
- Heterotrimeric stimulatory GTP-binding proteins (Gas); cAMP signaling system; cisplatin; radiation; DNA repair; apoptosis; XIAP; XRCC1; Epac; ATM; cervical cancer; lung cancer
- 학위논문 (박사)-- 서울대학교 대학원 : 의학과 생화학 전공, 2013. 2. 전용성.
- Heterotrimeric stimulatory GTP-binding proteins (Gas) activate cAMP signaling system and play important roles in the regulation of cell growth, proliferation, differentiation, and apoptosis. However, the role of Gas protein in DNA damage response has not been studied extensively. Thus, the role of Gas protein on DNA damage responses such as DNA repair and apoptosis was studied in this experiment.
In the first part of this study, in order to elucidate the molecular mechanism for Gas system to regulate apoptosis, the effect of the Gas system on cisplatin-induced apoptosis and the expression of IAP family proteins were examined in uterine cervical cancer cells. HeLa cells stably expression constitutively active GasQL were established by transfection. Stable expression of constitutively active Gas (GasQL) decreased the release of cytochrome c from the mitochondria to the cytosol and cleavage of caspase-3 and poly (ADP-ribose) polymerases in HeLa cells treated with 30 uM cisplatin, indicating that Gαs inhibited cisplatin-induced apoptosis. Treatment with forskolin also inhibited apoptosis of C33A and CaSKi cervical cancer cells. Expression of GasQL increased the expression of the X-linked inhibitor of apoptosis protein (XIAP) and partially maintained increased XIAP after cisplatin treatment. Knockdown of XIAP by siRNA augmented apoptosis. Expression of GasQL increased XIAP mRNA
this increase was inhibited by a protein kinase A inhibitor and cAMP response element (CRE) decoy. A cAMP response element (CRE)-like element at -1396 bp in the XIAP promoter was found to mediate the induction of XIAP by Gas. In addition, expression of GasQL protected against the ubiquitin/proteasome-dependent degradation of the XIAP protein. In conclusion, this study shows that Gas inhibits cisplatin-induced apoptosis by increasing transcription of XIAP and by decreasing degradation of XIAP protein in HeLa cervical cancer cells.
In the second part of this study, to verify the hypothesis that the cAMP signaling may modulate DNA repair activity, the effects of the cAMP signaling system on r-ray-induced DNA damage repair was investigated in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GasQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GasQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after r-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following r-ray irradiation. From these results, it was concluded that the cAMP signaling system inhibits the repair of r-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.
In the third part of this study, the mechanism through which cAMP signaling regulates activation of ATM, which is the master regulator of DNA damage responses, and cellular responses to ionizing radiation was investigated. Transient expression of constitutively active stimulatory G protein (GasQL) significantly inhibited the radiation-induced phosphorylation of ATM in H1299 human lung cancer cells. Treatment with okadaic acid abolished the inhibitory effect of Gas on the radiation-induced ATM phosphorylation. Expression of GasQL increased the phosphorylation of PP2A B56dsubunit and PP2A activity, and this PP2A-activating effect of Gas was completely removed by H89. Expression of GasQL increased radiation-induced cleavage of caspase-3 and PARP and the number of early apoptotic cells, and treatment with KU55933 also increased the apoptosis. Treatment of B16-F10 mouse cells with forskolin increased the radiation-induced phosphorylation of B56d but decreased the radiation-induced phosphorylation of ATM in the mouse lung. Expression of GasQL increased the IkBa protein and decreased the levels of p50 and p65 subunits of NF-kB in nucleus after r-ray irradiation, and PDTC increased the radiation-induced apoptosis. Pretreatment with prostaglandin E2 or isoproterenol increased the phosphorylation of B56d and decreased the radiation-induced ATM phosphorylation and increased the apoptosis. In conclusion, cAMP signaling inhibits radiation-induced ATM activation by PKA-dependent activation of PP2A, and this signaling mechanism augments radiation-induced apoptosis by reducing ATM-dependent activation of NF-kB in lung cancer cells and mouse lung tissue.
From these studies, Gas protein/cAMP signaling system regulates DNA damage responses such as DNA damage repair and apoptosis by various mechanisms. These findings suggest that Gas protein/cAMP signaling system could be targeted to enhance the therapeutic efficiency of various cancers.