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author:("Zhang, yingzi")
1.  Caspase-2 Deficiency Enhances Aging-Related Traits in Mice 
Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.
doi:10.1016/j.mad.2006.11.030
PMCID: PMC1828128  PMID: 17188333
caspase-2; maximum lifespan; bone; hair growth; fat
2.  A Hybrid System Using Both Promoter Activation and Gene Amplification for Establishing Exogenous Protein Hyper-Producing Cell Lines 
Cytotechnology  2003;43(1-3):11-17.
We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10−6 cells day−1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml−1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.
doi:10.1023/B:CYTO.0000039901.92984.7a
PMCID: PMC3449599  PMID: 19003202
CHO cells; gene amplification; glutamine synthetase (GS); promoter-activated production (PAP) system; ras oncogene; recombinant protein production
3.  A regulatable selective system facilitates isolation of heterologous protein hyper-producing mammalian cells without gene amplification 
Cytotechnology  2002;40(1-3):13-22.
In this article, we describe a new method that facilitates to isolate mammalian cells inducible hyper-producing heterologous proteins. This method uses the tetracycline-inducible system to express both the selection marker and the heterologous gene, therefore, allows to increase the selection pressure by reducing the transcription of the selection maker gene. Using this method, we were able to isolate recombinant Chinese hamster ovary cells with a high efficiency. One of established clones produced the recombinant bovine β-lactoglobulin as heterologous protein at a peak rate of 12 μg 10-6 cells/day with an inducibility of about 100-fold. This clone was over expressed them RNA of β-lactoglobulin and the drug resistant gene but did not amplify their genes. When cultured in a hollow fiber bioreactor, the cells were able to secrete β-lactoglobulinover 300 μg ml-1. This method is applicable to a broad range of eukaryotic systems and is of general value to technology for recombinant protein production.
doi:10.1023/A:1023945517446
PMCID: PMC3449528  PMID: 19003100
CHO cells; doxycycline; recombinant protein production; selection pressure; tetracycline-inducible system
4.  Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells 
Cytotechnology  2001;35(1):9-16.
We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The λ light chain(C5λ) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5λ hyper-producing cell line by transfecting ras cloneI with the C5λ gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5λ andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5λ protein, which might be caused by that the amount of produced C5λ in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.
doi:10.1023/A:1008179919857
PMCID: PMC3466615  PMID: 19003276
cytomegalovirus promoter; HB4C5; mass production; OAP system; oncogene
5.  Efficient and inducible production of human interleukin 6 in Chinese hamster ovary cells using a novel expression system 
Cytotechnology  1997;25(1-3):53-60.
High level and inducible production of human interleukin 6 (hIL-6) was achieved using a novel expression system in Chinese hamster ovary (CHO) cells. In this system, the transcription of hIL-6 gene under the control of PhCMV*-1 promoter composed of tetracycline operator sequences and a minimal promoter is activated by a chimeric transactivator (tTA) composed of tetracycline repressor and transactivating domain of VP16 protein of herpes simplex virus. The transcription of tTA gene, which is also under the control of PhCMV*-1 promoter, is activated by itself via a positive feedback cycle. The expression of both genes is further enhanced by potentiating the VP16 transactivating domain of tTA transactivator with pX protein of hepatitis B virus. In the presence of tetracycline, the tTA transactivators can not bind to PhCMV*-1 promoter, therefore, the expression of hIL-6 and tTA gene is suppressed, and the pX will not activate basal transcription. In the absence of tetracycline, tTA transactivators bind to PhCMV*-1 promoter and activate efficient transcription of hIL-6 and tTA gene, and the transcription is further enhanced by pX via VP16 transactivating domain. Using this strategy, we isolated a clone (UX1) producing hIL-6 at a rate about 1425 ng/106 cells/day. Furthermore, the hIL-6 production is stringently regulated by tetracycline. This results suggested a novel strategy to establish highly efficient, inducible and cell type independent recombinant protein production system by using an artificial promoter to recruit transactivators and coactivators which can synergistically activate transcription.
doi:10.1023/A:1007972002180
PMCID: PMC3466749  PMID: 22358879
CHO cells; coactivator; recombinant protein production system; transactivator
6.  Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells 
Cytotechnology  1997;23(1-3):193-196.
The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.
Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.
doi:10.1023/A:1007955332526
PMCID: PMC3449871  PMID: 22358535
Chinese hamster ovary cell; human cytomegalovirus promoter; insulin; phosphatidylcholine-specific phospholipase C; signal transduction

Results 1-6 (6)