Premium
Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G 1 , but Before the G 1 /S Transition, Involves Polypeptides
Author(s) -
Langan Thomas J.,
Slater Mary C.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02029.x
Subject(s) - cycloheximide , biology , glial fibrillary acidic protein , microbiology and biotechnology , thymidine , dna synthesis , bromodeoxyuridine , cell cycle , cell culture , in vitro , biochemistry , protein biosynthesis , cell , cell growth , immunology , genetics , immunohistochemistry
The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum‐depleted (0.1%, vol/vol) for 48 h on days 4–6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70–80% of the cells were glial fibrillary acidic protein (GFAP)‐negative by indirect immunofluorescence; 79 ± 7% were GFAP‐positive after an additional 3 days. Serum shift‐up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 ± 6% to 75 ± 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 ± 6 versus 380 ± 32 cpm/μg of protein/h of [ 3 H]thymidine uptake). Addition of mevalonate (25 m M ) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 μ M ), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G 1 /S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine‐labeled nuclei increased from 0% to 75 ± 9%, and DNA synthesis increased 10‐fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G 1 , but before the G 1 /S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.