Expression of the transcription factor GADD153 is an indicator of apoptosis for recombinant chinese hamster ovary (CHO) cells.

Loss of cell viability, through engagement of apoptotic cell death, represents a limitation to maintenance of high levels of productivity of recombinant animal cells in culture. The ability to monitor the status of recombinant cells, and to define indicators of their "well-being," would present a valuable approach to permit a rational intervention at appropriate times during culture. Growth arrest and DNA damage gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors and has been associated with apoptosis. We have examined the expression of GADD153 in conditions associated with apoptosis of recombinant CHO cells in batch culture. GADD153 expression is very low in CHO cells growing in the exponential phase of batch culture but is activated as cells enter the decline phase. Depletion of nutrients (glucose or glutamine) causes activation of GADD153 expression as does the imposition of endoplasmic reticulum stress. In all cases, there is a good relationship between the extent of apoptosis that occurs in response to each stress and the degree of GADD153 expression. In addition, nutrient refeeding or reversal of stress produces a concomitant decrease in expression of GADD153 and the susceptibility to apoptosis. Thus, GADD153 appears to offer a valid indicator of apoptosis and illustrates the potential for definition of monitors of cellular status related to the likelihood of apoptosis of cell populations.

Expression of CCAAT/enhancer binding protein family genes in monolayer and sandwich culture of hepatocytes: induction of stress-inducible GADD153.

Removal of hepatocytes from their physiological environment for experimentation in vitro activates loss of liver-specific phenotype. Hepatocytes cultured in a sandwich configuration reportedly maintain greater expression of certain liver-specific genes than hepatocytes in monolayer cultures. We show that sandwich culture of rat hepatocytes improves retention of expression of a liver-enriched transcription factor, C/EBPalpha (CCAAT/enhancer binding protein alpha), which regulates many liver-specific genes. However, we also demonstrate increased expression of a stress-responsive C/EBP homologue, GADD153 (growth arrest and DNA damage gene 153), during monolayer culture, which may promote dedifferentiation. Induction of GADD153 was not prevented in sandwich cultured hepatocytes. Activation of a homologue of the mouse GADD153 target gene, doc1, was observed in monolayer and sandwich culture, suggesting that GADD153 was transcriptionally active. We suggest that the capability of sandwich cultures to maintain hepatocyte phenotype may be limited by the altered profile of transcription factor activity.

Characterization of the stability of recombinant protein production in the GS-NS0 expression system.

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.

Protein kinase C isoenzyme expression in retinal cells.

Protein kinase C (PKC) is involved in both the physiological and pathophysiological processes in the retina and plays an important role in signal transduction. The aim of this study was to determine the PKC isoenzyme profile in three retinal cell types in culture, namely RPE cells, pericytes and retinal microvascular endothelial cells. Confluent cultures were lysed and isoenzyme expression detected by Western blotting. PKC isoenzymes alpha, beta(2) and delta were observed for all three cell types while beta(1) was specific for RPE cells. This study has characterised the PKC isoenzyme profile in three retinal cell types and suggests that defining the cell-specific isoenzyme pattern is an important step in understanding their precise physiological role and regulation in the retina.

Advances in animal cell recombinant protein production: GS-NS0 expression system.

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

Hepatocyte isolation stimulates formation of interferon stimulatory response element DNA-protein complexes.

We have examined the relationship between intracellular signalling pathways and loss of differentiated function during hepatocyte isolation and culture. We have shown that isolation induces the activation of the interferon stimulatory response element (ISRE). This activation was transient and peaked at 3 h before it returned to basal by 24 h of culture. Interferon regulatory factor-1 (IRF-1) was shown to be important for generation of ISRE complexes by electromobility shift assays and supershift intervention. IRF-1 was translocated to the nucleus in parallel with changes to ISRE complex formation. The p38 kinase inhibitor, SB 203580, diminished the formation of ISRE binding complexes. Hence p38 kinase may be involved in the activation and binding of IRF-1 or related proteins to the ISRE motif. Changes in ISRE activation levels in cultured hepatocytes may have important implications in primary hepatocyte differentiation and loss of function.

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression.

Gene 33 is a putative immediate early gene and we have shown that mRNA encoding for gene 33 exhibits a transient increase as a result of the procedures used for hepatocyte isolation. The stress-activated protein kinases p46 JNK, p54 JNK, and p38 SAPK are activated by hepatocyte isolation and precede changes in gene 33 mRNA content. Although each SAPK isoform shows a distinctive profile of activity during isolation and subsequent hepatocyte culture, in each case the activation is transient and is largely reversed within 3 h of hepatocyte isolation. SB 203580, a p38 SAPK inhibitor, prevents the change to gene 33 expression in response to hepatocyte isolation. Given the possible role of gene 33 as an immediate early gene, the data presented here have general implications for control of hepatocyte proliferation and differentiation.

Rat primary hepatocytes and H4 hepatoma cells display differential sensitivity to cyclic AMP at the level of expression of tyrosine aminotransferase.

We have shown that the sensitivity of isolated hepatocytes and H4 hepatoma cells to cyclic AMP is different. In terms of activation of tyrosine aminotransferase at mRNA and activity level in response to cyclic AMP, isolated hepatocytes are 10-fold more sensitive. Hepatocytes and H4 hepatoma cells show similar sensitivities to cyclic AMP at the level of protein kinase A activation and phosphorylation of cyclic AMP response element binding protein (CREB) and the differential sensitivity must reside at other sites. The consequences of these findings to studies of control phenomena at the transcriptional level is discussed.

Apoptosis regulation and its applications to biotechnology.

Increased knowledge of the molecular interactions that characterize the control of apoptosis is identifying possible novel approaches to inhibit cell death and generate enhanced viable cell biomass. The application of such approaches to recombinant mammalian cells promises to improve the cost-effectiveness of industrial-scale protein expression.

Dichloroacetate inhibits glutamine oxidation by decreasing pyruvate availability for transamination.

We have shown that dichloroacetate (DCA) inhibits growth, glutamine oxidation, and pyruvate and alanine production in a concentration-dependent manner in PQXB 1/2 hybridoma cells. The use of inhibitors indicates that glutamine oxidation proceeds by an aminooxyacetate-sensitive transamination reaction in this cell line. Addition of pyruvate to DCA-treated cells restored glutamine oxidation to control values. Our data suggest that DCA inhibits glutamine oxidation by decreasing the availability of pyruvate for transamination, which in turn results in glutamate accumulation and a consequent inhibition of glutaminase activity. Impaired glutamine catabolism in the presence of DCA has subsequent effects on overall metabolic balance and cell maintenance and growth.

NSO myeloma cell death: Influence of bcl-2 overexpression.

The productivity of recombinant mammalian cell lines growth in batch culture is often limited by the rapidity with which cells die on entry into the decline phase (the period of culture after the maximum cell density has been reached and where cell viability begins to fall). We examined the decline phase characteristics of the NSO myeloma cell line with a view to modulating the cell death that ensues. Examination of nuclear morphology during culture revealed that the onset of the decline phase was marked by a time-dependent increase in the percentage of cells that exhibited condensed and fragmented nuclei. Furthermore, these changes coincided with a fall in DNA integrity. High molecular weight DNA appeared to be degraded into oligonucleosomal fragments. Taken together, these observations indicated that NSO cells die by the process of apoptosis. The protein encoded by the bcl-2 gene has been shown to counter apoptosis induced by a large variety of stimuli and in a number of different cell types, but is not expressed in NSO cells. We examined whether overexpression of this protein could prevent/delay the onset of cell death seen during batch culture and also in response to serum limitation. Bcl-2 failed to affect the decline phase characteristics and serum dependence of NSO cells. In our search to explain these findings, we found that the NSO cell line expresses bax and also a high level of another Bcl-2 related protein, Bcl-x(L). Given that Bcl-X(L) is a sequence and functional homologue of Bcl-2, it is possible that Bcl-2 is redundant in the NSO cell background. These data therefore indicate that cells such as NSO, which are used in biotechnologically important processes such as generation of hybridomas and expression of recombinant proteins, may express only a subset of genes important in apoptotic regulation. Modulation of the death characteristics of such cells will need to take account of the expression profile of such genes and their regulatory interactions.

Dichloroacetate increases cell and antibody yields in batch cultures of a hybridoma cell line.

We have studied the effect of the pyruvate dehydrogenase (PDH) activator, dichloroacetate (DCA), on the growth, metabolism, and productivity of the PQXB (1/2) hybridoma cell line. In control batch cultures, cessation of growth and the onset of decline phase coincided with the time at which the media became exhausted of glutamine. Supplementation of the media with DCA (1 mM) extended the growth phase of this cell line by approximately 20 h without affecting its growth rate. This prolonged period of growth resulted in an increased maximum cell density (16%) and final antibody yield (55%). Repeat experiments showed these effects to be reproducible, with the increases in antibody yield being between 50 and 60%. DCA did not affect the specific rates of glucose utilization and lactate production. However, it decreased the specific glutamine consumption rate. This characteristic of DCA action appeared, at least in part, to provide an explanation for the extended growth phase exhibited by DCA-treated cultures, since it delayed the time at which the media became depleted of glutamine. The consumption and production kinetics for various nutrients and their metabolites in both control and DCA-treated cultures suggested that: (1) glutamine catabolism proceeded by a pathway involving conversion to glutamate by glutaminase followed by subsequent transamination by alanine aminotransferase, and (2) DCA decreased the specific glutamine consumption rate by directly or indirectly inhibiting the transamination. It is expected that the routine inclusion of DCA in media used for hybridoma cultivation will be valuable for enhancement of monoclonal antibody (Mab) yields on a laboratory scale. (c) 1996 John Wiley & Sons, Inc.

Inhibitors of signalling identify differential control processes responsible for selective effects of insulin on the expression of phosphoenolpyruvate carboxykinase and gene 33 in rat H4 hepatoma cells.

Gene 33 and phosphoenolpyruvate carboxykinase (PEPCK) present excellent model systems for the analysis of the differential control of hepatic gene expression by the insulin-regulated signal-transduction pathway(s). We have analysed the importance of specific components in the insulin-regulated transduction pathway(s) towards enhanced gene expression (gene 33) and inhibited gene expression (PEPCK) by examination of the influence of selective inhibitors. Rapamycin, which inhibits the 70 kDa S6 kinase (p70rsk) does not influence the actions of insulin on gene 33 or PEPCK; thus the kinase p70rsk appears to play no direct role in the regulation of expression of these two hepatic genes. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, differentiates between processes involved in insulin regulation of gene 33 and PEPCK mRNA expression. Although the actions of insulin on gene 33 expression are abolished by wortmannin, the actions of insulin on PEPCK expression are insensitive to wortmannin. The existence of wortmannin-sensitive and rapamycin/wortmannin-insensitive pathways for transducing insulin signals to factors controlling gene expression, and the differential actions on specific genes, presents an initial step towards deciphering the linkage between signalling components and selective control of gene expression.

Regulation of tyrosine aminotransferase activity by glucagon and cAMP analogues in chick embryos in ovo.

Glucagon, dibutyryl cAMP (Bt2cAMP) and 8-(4-chlorophenylthio) cAMP (CptcAMP), singly or when combined, stimulated tyrosine aminotransferase (TAT) activity in 17-day-old chick embryos in ovo. Maximal induction was produced within 4 hr of injection of the inducers. The effects of glucagon and the cAMP analogues were not additive. Glucagon administration was accompanied by a rapid increase in hepatic cAMP concentration which remained elevated for at least 4 hr. The stimulated increase in TAT activity elicited by the hormone or cyclic nucleotide was prevented by injection of cycloheximide or cordycepin. These results are discussed vis-à-vis the possible regulation of TAT in ovo by physiological concentrations of glucagon and the likely role of cAMP as a second messenger in this process during chick embryogenesis.

Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1.

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.