Effect of Akt overexpression on apoptosis and autophagy in cChinese hamster ovary cells = Akt의 과발현이 CHO 세포에서 apoptosis와 autophagy에 미치는 영향

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Over the past decades, Chinese hamster ovary (CHO) cells became the most widely used mammalian cells for high-level expression of foreign therapeutic proteins ranging from antibodies to growth factors in industry. It is well-established that, when the most nutrients are deprived during batch culture, cells lose their viability and eventually die through the complex mechanisms of programmed cell death (PCD). Especially, apoptosis and autophagy were investigated. In this study, we have used two different antibody producing CHO cell lines, Ab1 and Ab2. Two cell lines were cultivated in different media containing high (25.0 mM glucose and 4.0 mM glutamine) or low (5.6 mM glucose and 2.0 mM glutamine) nutrient. Thus, they showed different growth profiles and antibody production profiles. However, both cell lines stopped proliferating and producing antibodies when the nutrient was deprived. We have shown that CHO cells were undergoing apoptotic and autophagic cell death towards the end of batch culture. Apoptotic cell death was demonstrated by chromosomal DNA fragmentation in addition to the cleavage of PARP protein, and autophagy was evidenced by the accumulation of 16 kDa form of LC3-II (an autophagosomal protein that is processed from 18 kDa to 16 kDa upon autophagy induction) and autophagic vacuoles. Since it is well known that anti-apoptotic CHO cell engineering is important for achieving a high-level protein production in large-scale culture, one of the major survival proteins, Akt (a serine/threonine kinase) was overexpressed in Ab1 and Ab2 cell lines. Akt is known to be activated by growth factors or cellular stresses and was found to function upstream of a major regulator of autophagy, mammalian target of rapamycin. Several studies showed that Akt activation inhibited cell death caused by apoptotic signal (Kauffmann-Zeh et al. 1997; Kennedy et al. 1997; Kulik et al. 1997). The constitutively active Akt (CA-Akt) was generated by Kohn et al., (Kohn et...
Advisors
Lee, Gyun Minresearcher이균민researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2007
Identifier
268698/325007  / 020025890
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2007. 8, [ xi, 138 p. ]

Keywords

Akt; Chinese hamster ovary cell; apoptosis; autophagy; programmed cell death; 생존 유전자; CHO 세포; 세포예정사; Akt; Chinese hamster ovary cell; apoptosis; autophagy; programmed cell death; 생존 유전자; CHO 세포; 세포예정사

URI
http://hdl.handle.net/10203/27635
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=268698&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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