Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium.
When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20 ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death - at 1.0 microg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.
Adamson
Lars
L
Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden.
Walum
Erik
E
D000826:Animal Testing Alternatives; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D016466:CHO Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020170:Caspase 1 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D016895:Culture Media, Serum-Free; D007004:Hypoglycemic Agents; D007328:Insulin; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D007334:Insulin-Like Growth Factor I; D020170:Caspase 1