[Effect of bcl-2 over-expression on the membrane structure of NG108-15 nerve cells: effectiveness of membrane-structure analysis using FACS for the first screening of apoptosis-inducing drugs].
Bcl-2 is a Bcl-2 family protein that is known to be anti-apoptotic and is predominantly localized to the mitochondria. We previously showed that an analgesic, buprenorphine hydrochloride (Bph), induces apoptosis in the rodent-derived nerve cell line, NG108-15, through the mitochondrial apoptotic route. A Bcl-2-overexpressing strain of NG108-15 cells, Bcl-2 (P2), was established, and the effect of Bcl-2 expression on Bph-induced apoptosis was compared between the mock vector-transfected NG108-15 cells and the Bcl-2 (P2) cells. The Bcl-2 (P2) cells died after treatment with Bph, and we observed all the biological and morphological markers of apoptosis that we tested for. In flow cytometric analysis, a difference in the cell membrane phospholipid flip-flop pattern-a feature of apoptosis- was observed between the NG108-15 cells and the Bcl-2 (P2) cells. Here, we show by flow cytometric analysis that Bcl-2 over-expression may affect the membrane structure of Bcl-2 (P2) cells. An increased fluorescein isothiocyanate (FITC) signal of annexin V-FITC, which typically represents phospholipid flip-flop of the cellular membrane in early apoptosis, was barely detected in the Bcl-2 (P2) cells. Since our previous study reported the localization of over-expressed Bcl-2 protein to the cell membrane of Bcl-2 (P2) cells, together these observations suggest that the Bcl-2 protein may affect the integrity of the structure of the NG108-15 cell membrane.
Kugawa
Fumihiko
F
Department of Biological Pharmaceutical Sciences, College of Pharmacy, Nihon University, Funabashi Chiba.
Ueno
Akemichi
A
Aoki
Masatada
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D002047:Buprenorphine / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000473:pathology*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D009474:Neurons / Q000166:cytology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology*
D019253:Proto-Oncogene Proteins c-bcl-2; D002047:Buprenorphine