In our early work the gene siat7e was identified to affect the adhesion and the morphology of Hela cells. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells resulted in greater aggregation and morphological changes. Based on the above work we were able to convert the anchorage dependent MDCK cells to anchorage independent cells by transfection with the human siat7e gene (ST6GalNac V). The converted cells were able to produce the influenza virus in bioreactors demonstrating their capability to replace the current egg based production process. Additional attempt to improve cellular properties of mammalian cells was based on identifying microRNAs that affect cells apoptosis. Microarray comparison of microRNAs in CHO cells exposed to fresh or depleted media revealed up-regulation of miR-297-669 cluster in CHO cells subjected to depleted media. miR-466h was chosen for further analysis. By implementing bioinformatics and experimental tools we predicted and verified the miR-466h anti-apoptotic targets. We continued this observation in three tracks. The first was the understanding of the mechanism associated with the activation of the apoptosis cascade as a result of the nutrient depletion, the second was to engineer cells that are more resistant to apoptosis and therefore can increase their growth and protein production properties, and the third was to conduct large scale screening of potential microRNAs that can inhibit apoptosis and their possible use in cancer research. The results of the first track showed that that the time-dependent activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation which was the result of glucose deprivation-induced oxidative stress. This oxidative stress caused the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity and increased acetylation in miR-466h-5p promoter region which led to the activation of this miRNA. In the second track we evaluated the effect of stable inhibition of mmu-miR-466h-5p in CHO cells on their ability to resist apoptosis onset and their production properties. The expression of mmu-miR-446h-5p in the engineered anti-miR-466h CHO cell line was significantly lower than in the negative control and the parental CHO cells. These engineered cells reached higher maximum viable cell density and extended viability compared with negative control and parental CHO cells in batch cell cultures which resulted in the 53.8 % and 41.6% increase of integral viable cells. The extended viability of anti-miR-466h CHO cells was the result of delayed Caspase-3/7 activation by more than 35 hours, and the increased levels of its anti-apoptotic gene targets (smo, stat5a, dad1, birc6, and bcl2l2) to between 2.1- and 12.5-fold compared with the negative control CHO in apoptotic conditions. The expression of secreted alkaline phosphatase (SEAP) increased 43% and the cell-specific productivity increased 11% in the stable pools of anti-miR-466h CHO compared with the stable pools of negative control CHO cells. The above results demonstrate the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression. In the third track we conducted large-scale screening of the complete miRNA mimics library demonstrated that hsa-miR-15a-3p had a pro-apoptotic role in the following human cancer cells: HeLa, AsPc-1, MDA-MB-231, KB3, ME180, HC T-116 and A549. MiR-15a-3p is a novel member of the pro-apoptotic miRNA cluster, miR-15a/16, which was found to activate Caspase-3/7 and to cause viability loss in B/CMBA.Ov cells during preliminary screening. Subsequent microarrays and bioinformatics analyses identified the following four anti-apoptotic genes: bcl2l1, naip5, fgfr2 and mybl2 as possible targets for the mmu-miR-15a-3p in B/CMBA.Ov cells. Follow-up studies confirmed the pro-apoptotic role of hsa-miR-15a-3p in human cells by its ability to activate Caspase-3/7, to reduce cell viability and to inhibit the expression of bcl2l1 (bcl-xL) in HeLa and AsPc-1 cells. MiR-15-3p was also found to reduce viability in HE K293, MDAMB-231, KB3, ME180, HC T-116 and A549 cell lines and, therefore, may be considered for apoptosis modulating therapies in cancers associated with high Bcl-xL expression (cervical, pancreatic, breast, lung and colorectal carcinomas). The capability of hsa-miR-15a-3p to induce apoptosis in these carcinomas may be dependent on the levels of Bcl-xL expression. The use of endogenous inhibitors of bcl-xL and other anti-apoptotic genes such as hsa-miR-15a-3p may provide improved options for apoptosis-modulating therapies in cancer treatment compared with the use of artificial antisense oligonucleotides.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$357,416
Indirect Cost
City
State
Country
Zip Code
Chu, Chia; Bottaro, Donald P; Betenbaugh, Michael J et al. (2016) Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells. PLoS One 11:e0148075
Gallagher, Emily J; LeRoith, Derek; Stasinopoulos, Marilyn et al. (2016) Polyol accumulation in muscle and liver in a mouse model of type 2 diabetes. J Diabetes Complications 30:999-1007
Xiao, Su; Chen, Yu Chi; Buehler, Eugene et al. (2016) Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol Bioeng :
Kumar, Amit; Shiloach, Joseph; Betenbaugh, Michael J et al. (2015) The beta-3 adrenergic agonist (CL-316,243) restores the expression of down-regulated fatty acid oxidation genes in type 2 diabetic mice. Nutr Metab (Lond) 12:8
Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard (2015) Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1. Methods Mol Biol 1272:51-64
Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel et al. (2015) Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics. J Proteome Res 14:4687-703
Kumar, Amit; Baycin-Hizal, Deniz; Shiloach, Joseph et al. (2015) Coupling enrichment methods with proteomics for understanding and treating disease. Proteomics Clin Appl 9:33-47
Xiao, Su; Chen, Yu-Chi; Betenbaugh, Michael J et al. (2015) MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells. Biotechnol Bioeng 112:1632-43
Heffner, Kelley M; Hizal, Deniz Baycin; Kumar, Amit et al. (2014) Exploiting the proteomics revolution in biotechnology: from disease and antibody targets to optimizing bioprocess development. Curr Opin Biotechnol 30C:80-86
Kumar, Amit; Harrelson, Thomas; Lewis, Nathan E et al. (2014) Multi-tissue computational modeling analyzes pathophysiology of type 2 diabetes in MKR mice. PLoS One 9:e102319

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