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저산소상태 및 RNA 간섭기법을 이용한 Adenylate Kinase 4 (AK4)의 분자 생리적 기능규명

Title
저산소상태 및 RNA 간섭기법을 이용한 Adenylate Kinase 4 (AK4)의 분자 생리적 기능규명
Other Titles
Studies on the molecular physiology of Adenylate Kinase 4 (AK4) by using hypoxia and RNA interference technology
Author
콩판즈
Alternative Author(s)
Kong, Fanzhi
Advisor(s)
김효준, Bert Binas
Issue Date
2012-08
Publisher
한양대학교
Degree
Doctor
Abstract
Adenylate kinases (AK, E.C. 2.7.4.n), which catalyze the nucleotide phosphoryl exchange reaction MgNTP+AMP ↔ MgNDP+ADP (N = A or G), are known for their important roles in nucleotide homeostasis. Within the AK family, AK isozyme 4 (AK4) shows the lowest, almost negligible, phosphoryl transferase activity in vitro, and it was proposed that AK4 plays a non-enzymatic role. AK4 expression increases under hypoxia stress in several tissues in vivo and in all previously tested cell lines in vitro; however, the biological meaning of this observation remains unclear. Here, we confirmed a previous observation that AK4 expression is increased in hypoxic human embryonic kidney (HEK293) cells, but in addition we discovered that human hepatocellular carcinoma (HepG2) cells show an unusually high basal AK4 level and uniquely downregulate, rather than increase, AK4 under hypoxia. In order to understand the biological meaning of this discrepancy, we compared the effects of hypoxia and shRNA-mediated AK4 knockdown on HEK293 and HepG2 cells. To this end, we assessed the global transcription profiles (by microarrays), the expression of selected genes (by qRT-PCR or Western blotting), cell numbers (by counting), cell survival (by Trypan blue exclusion or MTT assay), the degradation of DNA (by agarose gel electrophoresis), and the cell cycle parameters (by flow cytometry). HEK293 and HepG2 cells showed a dramatically different response to hypoxia not only with respect to AK4 expression but also with respect to more global physiological events. Hypoxic HEK293 cells maintained their viability and showed no impairment of proliferation, and global transcription profiling revealed a net increase of cell cycle- and protein synthesis-related genes, but a net decrease of apoptosis-related genes in hypoxic HEK293 cells. By contrast, hypoxic HepG2 cells showed a significantly reduced viability and impaired proliferation, and their gene expression profile was shifted in a manner reciprocal to that just described for HEK293 cells. Among the apoptosis-related genes, the most prominent one showing upregulation in hypoxic HepG2 cells was MCL1S, as confirmed by qRT-PCR. We therefore wondered whether AK4 is playing similar roles in these cells. Lentiviral shRNA-mediated AK4 knockdown lead to a decreased proliferation rate of both HEK293 cells (as previously published) and HepG2 cells. HepG2 cells also showed a reduced viability and clonogenicity, similar to previous results published on HEK293 cells. Hence, AK4 has principally similar biological roles in HEK293 and HepG2 cells. However, there were interesting differences in the details. Notably, cell cycle parameters were undisturbed by the knockdown in HEK293 cells, but showed a significant increase of the subG1 fraction and decrease of S phase fraction in HepG2 cells. Furthermore, HepG2 but not HEK293 cells showed a massive increase of DNA degradation due to the knockdown. Taken together, our results show that (i) HEK293 cells and HepG2 cells use different strategies to deal with hypoxic stress, (ii) yet AK4 fulfills principally similar functions in these cells. We can further conclude that (iii) AK4 is subordinated under the stress response strategy rather than being a master regulator of the stress response. And finally, (iv) our data lead to the proposition that in AK4-expressing cells types, AK4 levels indicate the level of basal stress. According to this proposition, it may be a higher level of basal stress perceived by HepG2 cells that leads to their high basal AK4 level and consequently to a defeatist stress response strategy, while HEK293 cells can afford a more active survival strategy.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/136419http://hanyang.dcollection.net/common/orgView/200000420500
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > DEPARTMENT OF BIOCHEMISTRY(생화학과) > Theses (Ph.D.)
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