title: BCL-2 and MCL-1 expression in Chinese hamster ovary cells inhibits intracellular acidification and apoptosis induced by staurosporine.
abstract: Multiple physiological and pharmacological stimuli induce cells to die by apoptosis. In many cases, this apoptosis is inhibited by BCL-2, suggesting the involvement of a common regulatory pathway. One frequent characteristic of apoptosis is the digestion of DNA into oligonucleosome-length fragments. Intracellular acidification and increased intracellular calcium have been variously implicated in activating the endonuclease responsible for this DNA digestion. To explore the involvement of these potential signals in endonuclease activation, we have analyzed three Chinese hamster ovary cell lines: a parental line, one expressing a cDNA encoding BCL-2, and the third expressing the BCL-2 family member MCL-1. Apoptosis was induced with the protein kinase inhibitor staurosporine and intracellular pH and calcium were measured by flow cytometry. We found that both MCL-1 and BCL-2 inhibited DNA digestion compared to the parent cell line, although BCL-2 was more potent in this regard. Concurrent with DNA digestion, we observed intracellular acidification; MCL-1 and BCL-2 inhibited intracellular acidification to an extent commensurate with their ability to inhibit DNA digestion. In contrast, staurosporine caused a dose-dependent increase in intracellular calcium in all three cell lines, demonstrating that intracellular free calcium levels did not correlate with the induction of apoptosis. These results suggest that BCL-2 and MCL-1 may regulate a pathway for intracellular pH homeostasis during apoptotic cell death.
authors.affiliation: Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Reynolds
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: R W
authors.initials: RW
authors.lastname: Craig
authors.forename: A
authors.initials: A
authors.lastname: Eastman
mesh_terms: D000143:Acids / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001562:Benzimidazoles; D016466:CHO Cells / Q000166:cytology / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D019311:Staurosporine
chemical_list: D000143:Acids; D000470:Alkaloids; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011505:Protein-Tyrosine Kinases; D019311:Staurosporine; C017807:bisbenzimide ethoxide trihydrochloride; D002118:Calcium