| GADD45 (growth arrest and DNA damage-inducible gene 45)αis one of the growth arrest and DNA damage-inducible (Gadd) 45 gene family members, which can play an important role in cell cycle arrest, DNA repair, senescence and apoptosis when activated by a variety of DNA damage signals, such as UV, methylmethanesulfonate (MMS), ionizingradiation (IR) and so on. Therefore, GADD45αis well-accepted as a potential tumor suppressor. However, a recently published report has demonstrated the direct involvement of GADD45αin tumor cell survival. And the evidences from the clinical researches have illustrated the correlation between higher GADD45αexpression and enhanced tumor progression, radiotherapy resistance and lower survival rate of the patients. Therefore, the role of GADD45αin tumor cell survival, progression and apoptosis is still controversy and needs further investigation.In this study, we have investigated the role of GADD45αin mediating the apoptotic effect in the human hepatoma cell line, HepG2, under the exposure of arsenite, the effective tumor therapeutic agent. And the results show that arsenite induces the apoptosis of HepG2 cells accompanying with the persistent and strong induction of GADD45α. When arsenite-induced GADD45αexprssion was inhibited by its specific shRNA, cell apoptosis decreased dramatically under the same conditions, which results indicate a critical role of GADD45αin mediating arsenite-induced apoptosis in the HepG2 cells. Further investigation reveals that JNK/AP-1 pathway is the downstream target of GADD45αin response to arsenite exposure. In addition, c-Jun and Fra-1 are the major AP-1 components that are involved in arsenite -induced apoptosis in the HepG2 cells. Interestingly, signaling events relating to GADD45a/JNK/AP-1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure conditions. Our results thus have illustrated the selective pro-apoptotic role of arsenite in the hepatoma cells by activating GADD45a/JNK/AP-1 cell death pathway whereas with little effect on the normal hepatic cells.The expression of GADD45αcan be regulated at multiple levels, including transcriptional, post-transcriptional, translational and post-translational mechanisms. One of our recent researches have firstly demonstrated the constitutive ubiquitination and proteasome-dependent degradation of GADD45αin the resting normal diploid cells and the prevention of ubiquitination-dependent degradation and the accumulation of GADD45αunder the stress conditions. Therefore, after illustrating the downstream signal transduction pathways of GADD45αin mediating arsenite-induced apoptosis in the HepG2 cells in the first part, we step to investigate the upstream signaling events regulating arsenite-induced GADD45αexpression in the second part. We have also found the constitutive ubiquitination and proteasome-dependent degradation of GADD45αin the HepG2 cells. Arsenite exposure induces the accumulation of GADD45αby up-regulating its protein stability, instead of the transcriptional induction of the GADD45αgene. In order to reveal the mechanism involving in regulating GADD45αprotein stability, the three-dimensional structure of GADD45αis predicted under the aid of the bioinformatical software and then a series of GADD45αmutants are constructed to determine the amino acid(s) that are critical for regulating the ubiquitination and degradation of GADD45α. By comparing the difference of the half-lives between wt-GADD45αand the mutants, we have identified that two amino acid regions are important in regulating the ubiquitination of GADD45αdue to the extremely higher stability of the according mutants. E3 ubiquitin ligase plays a critical role in triggering the ubiquitination and degradation of the specific target protein. To identify the specific E3 ubiquitin ligase for GADD45α, yeast two-hybrid screen is carried out and the GADD45α-interacting ribosomal protein S7 is selected. We further demonstrate that S7 contributes to maintaining the protein stability of GADD45αby suppressing its ubiquitination and degradation. Moreover, S7 also functions as the upstream signaling molecule in mediating arsenite-induced GADD45αexpression. The molecular events by which S7 regulates the protein stability of GADD45αunder resting and stress conditions are currently under investigation.IKK/NF-κB pathway plays a very important role in regulating cell survival and apoptosis under stress conditions. Our previous research in the normal diploid cells have disclosed that the role of GADD45αin mediating cell apoptosis is closely related to the function of IKKαand IKKβ, two catalytic subunits of IKK complex. Therefore, in the third part, we investigate the role of IKKαand IKKβin mediating arsenite-induced apoptosis in the HepG2 cells. The results show that the expressions of IKKαand IKKβwere dramatically down-regulated in the HepG2 cells following arsenite exposure. The similar phenomenon also can be found in other solid tumor cell lines induced by arsenite as well as other tumor therapeutic agents. Interestingly, arsenite exposure induces up-regulation of NF-κB activity, which event is clearly independent of IKKαand IKKβactivities. Further investigation on disclosing the mechanisms responsible for IKKαand IKKβprotein down-regulation have showed that arsenite inhibits the transcription of IKKβgene, which results to the down-regulation of IKKβexpression; while lysosomal protein degradation pathway is involved in the IKKαprotein down-regulation under the same arsenite exposure conditions.According to the results of this thesis, we have revealed the signal transduction pathway in mediating GADD45α-regulated apoptosis in the human hepatoma cell line under arsenite exposure; at the same time, we have identified the primary mechanism responsible for regulating the ubiquitination degradation and accumulation of GADD45αunder resting or arsenite exposure conditions. These date will be great helpful to further disclose the tumor suppressor role of GADD45α. |
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