G2 cell cycle arrest and apoptosis are induced in Burkitt's lymphoma cells by the anticancer agent oracin

AbstractThe cytotoxic effect of the new potential intercalating anticancer drug oracin was studied on Burkitt's lymphoma cell line that overexpressed bcl-2 (BL bcl-2) and a control transfectant without the bcl-2 gene (BL SV2). Oracin showed a marked cytostatic effect on both BL SV2 and BL bcl-2 cells. IC50, as measured by the MTT assay, was approx. 5-times greater for BL bcl-2 cells (5.0 μmol/l) than for BL SV2 cells (1.0 μmol/l). There was no significant increase in apoptosis after 24 h of treatment with oracin (1.0 μmol/l) in both cell lines. However, after 48 h from the removal of oracin in BL SV2 culture the levels of apoptotic and secondary necrotic cells increased to 20 and 37%, respectively. In contrast, BL bcl-2 cells treated in a similar manner showed only basal levels of apoptotic and secondary necrotic cells. Analysis of the cell cycle profiles showed a significant increase of S and G2/M phases of the cell cycle in both cell lines after 6 h of drug treatment (1.0 μmol/l). The cells were arrested in G2/M phase of the cell cycle after 24 h, with no significant changes in cell viability. After 72 h, the viable BL SV2 cells were still in G2/M, however, the viability of this culture had fallen to approx. 5%. Flow cytometry analysis of the DNA content revealed the presence of a `sub-G2' region, which represented the apoptotic cells. The BL SV2 cells died after 72 h while they were in the G2/M phase. Although the treated BL bcl-2 cells were similarly arrested in the G2/M phase, they nevertheless remained with a relatively high viability (68%)

Effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridomas during batch culture

In order to investigate the effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridoma cells, cells in different growth stages during a batch culture were fixed and stored at 4 and -20 °C, respectively. Flow cytometric analysis indicates that both fixation temperatures can be used in monitoring the changes in intracellular antibody content of the cells during a batch culture. However, it is better to fix and store the cells at -20 °C than 4 °C with regard to preservation of intracellular antibody and storage stability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42491/1/10542_2004_Article_BF00150897.pd

Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Glycosylation of an Immunoglobulin Produced From a Murine Hybridoma Cell Line: The Effect of Culture Mode and the Anti-Apoptotic Gene, bcl-2

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (¼(G2þ0.5 G1)/(G0þG1þG2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas. Biotechnol. Bioeng. 2007;97: 156–169. 2006 Wiley Periodicals, Inc. KEYWORDS: apoptosis; bcl-2; cell culture; IgG; glycosylation; galactosylation; hybridom