Expression of alkaline phosphatase by a B-cell hybridoma and its modulation during cell growth and apoptosis.
The 7TD1 B-cell hybridoma was found to spontaneously express alkaline phosphatase (ALP), an enzyme which is produced by splenic B lymphocytes once optimally activated. Determination of ALP levels during cell growth and departure to apoptosis showed fluctuations. Following a temporary increase within the first 24 h, enzyme expression was maintained at high levels during the early proliferation stage, and then declined from 3 to 4 days in mid-exponential phase to basal levels at day 6 when living cells were no longer detectable and the apoptotic process was completed. The protein synthesis inhibitor, cycloheximide (1 microg/ml), decreased ALP production while stimulating a strong apoptosis of 7TD1 cells, within 4 h. Aphidicolin (1 microg/ml) maintained ALP production and provoked a release of ALP activity into the surrounding medium; it also induced apoptosis, but with a 24 h delay. Quantification of apoptosis and ALP expression by flow cytometry, after simultaneous staining of DNA with Hoechst 33342 and ALP with naphthol AS-TR phosphate/Fast Red RC fluorescent reagent, revealed cell cycle modulation of ALP expression, its activity increasing as 7TD1 cells progressed from G1 phase into S and G2/M phases of the cell cycle in control as well as in drug-treated cells. Kinetics of drug-induced apoptosis and higher expression of ALP associated preferentially with active cell growth during the prevention stage of apoptosis suggested a possible link between cellular ALP expression and cell survival.
Souvannavong
V
V
CNRS-URA 1116, Institut de Biochimie, Université Paris-Sud, 91405 Orsay, France.
Lemaire
C
C
De Nay
D
D
Brown
S
S
Adam
A
A
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage; D005434:Flow Cytometry; D006825:Hybridomas / Q000276:immunology*
D016590:Aphidicolin; D003513:Cycloheximide; D000469:Alkaline Phosphatase