Effects of environmental factors and gene expression on mammalian cell growth and productivity

Cultured cells are used commercially to produce recombinant proteins and antibodies. Build-up of metabolic by-products (CO2, ammonia and lactate) inhibits cell growth and decreases intracellular pH (pH i). Several transporters are involved in pH; regulation in mammalian cells. The Na+/H+ exchangers (NHE) transport H+ ions from cells during acidification to increase pH i. Osmolytes, such as amino acids, are accumulated by cells to protect from hyperosmotic stress. Elevated CO2 causes an increase in osmolality when base is added to control extracellular pH (pHe). In this study we examined whether selected amino acids or over-expression of NHE1 would provide CHO cells with greater protection from elevated CO2. We also examined whether over-expression of NHE1 would provide protection for elevated ammonia or lactic acid. Amino acids are able to partially offset decreases in CHO cell growth and protein production during exposure to elevated pCO2. Expression of at least "normal" levels of NHE1 is necessary for CHO cell survival during exposure to 30 mM lactic acid without pH adjustment or to 20 mM NH4Cl with pH adjustment. However, there was no benefit of over-expressing the transporter on CHO cell growth during elevated CO2 and elevated osmolality.;Epstein-Barr virus (EBV) is a herpesvirus that infects human B lymphocytes and epithelial cells. The only two EBV factors required for stable replication and maintenance of the virus are the origin of replication, oriP, and the EBV latently expressed transcriptional activator, EBNA1. EBNA1 is also known to activate transcription from oriP-containing reporter plasmids in short-term assays (two to four days post-transcription). Using fusion proteins, I have examined if EBNA1 activates transcription from oriP-containing plasmids in cells that maintain them stably. My results indicate that it does not. A domain of EBNA1 (amino acids 65 to 89) was identified recently as the transcription activation region of EBNA1. I have examined whether this transcription activation was dependent upon the AT-hook region of EBNA1 and found it to be so. However, the AT hook region of EBNA1 could not be replaced by those from a cellular protein, HMGA1a, for transcription activation.