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M C Slater

Publications and source records attributed to M C Slater.

6 recordsLinked to original sources
PubMed1994

Novel relationships of growth factors to the G1/S transition in cultured astrocytes from rat forebrain.

The cell cycle encompasses the sequential events regulating cell division. In mammalian brain, initiation of astrocyte cycling is critical during development and injury. To investigate the timing of growth factor requirements as they commit to passing through the G1 phase, primary and secondary rat astrocytes were stimulated to enter the cycle after serum or growth factor deprivation. Bromodeoxyuridine immunofluorescence was used to monitor S phase nuclei after growth factor re-addition (at time 0). Cycle kinetics were identical whether quiescent cultures were exposed to 10% (vol/vol) calf serum, or to a defined medium containing fibroblast growth factor, insulin, and epidermal growth factor. The control point in late G1 that represents commitment to achieving the G1/S transition was identified by cycloheximide (CHX, 0.1 microgram/ml) addition. Sensitivity to cycle arrest by CHX disappeared at 9-10 h. In contrast, shift-down to growth factor-deficient medium arrested cell cycling virtually until G1/S (12 h). With selective exposure during late G1 (9-12 h), no single agent permitted cycle progression. However, any two agents enabled cycling, and complementary or synergistic effects were apparent. These requirements were identical in astroglia from newborn and long-term cultures. Thus, temporal dissociation exists between the processes of escape from CHX sensitivity and from requirements for growth factors, two recognized hallmarks of commitment to cycle progression. Furthermore, simultaneous presence of at least two growth factors is necessary at or near G1/S. Both findings distinguish astrocytes from several other cell types.

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PubMed1992

Astrocytes derived from long-term primary cultures recapitulate features of astrogliosis as they re-enter the cell division cycle.

We investigated whether the initiation of cell cycling by astrocytes after prolonged quiescence in long-term primary cultures is associated with immunocytochemical changes that characterize reactive astrogliosis. Primary cultures of newborn rat brain were maintained for greater than 2 months in a stable quiescent state. Partially synchronous transition through a single cell cycle was achieved by trypsinization and replating, and then after 2-3 days, by 48 h of serum depletion and serum shift-up to 10% (time 0). At time 0, the percentages of cells decorated by monoclonal antibodies specific for bromodeoxyuridine (BrDU) after a 2.5 h pulse, and for glial fibrillary acidic protein (GFAP) and vimentin (VIM) were respectively 8 +/- 2, 15 +/- 4 and 10 +/- 3. By 24 h (S phase), 64 +/- 7% of nuclei were (BrDU+), and percentages of (GFAP+) and (VIM+) cells were 19 +/- 4 and 87 +/- 12, respectively. Dual label immunofluorescence showed that greater than 75% of (GFAP+) cells were indeed (VIM+/GFAP+) at 24 h, and that the percentages of (VIM+), (BrDU+) and (VIM+/BrDU+) cells were equivalent for the duration of the first cell cycle (36-48 h). By 72-96 h, (VIM+) cells decreased to less than 10%, and (BrDU+) cells numbered 32 +/- 8%, while (GFAP+) cells increased to around 90%. Ran-2 immunofluorescence at 96 h identified virtually all of the cells as type 1 astrocytes. Thus, astrocytes after months of quiescence give rise to cells that recapitulate a VIM/GFAP transition in a manner resembling astrogliosis, and do so in relation to progression through a single cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Quiescent astroglia in long-term primary cultures re-enter the cell cycle and require a non-sterol isoprenoid in late G1.

Astroglia proliferate during brain growth, and can divide again later, particularly during astrogliosis. We investigated whether astroglia in primary cultures of newborn rat brain similarly achieve a state of prolonged quiescence which enables re-entry into the cell division cycle. In cultures after 2 months, cell number plateaued and there were sharp decreases in [3H]thymidine incorporation (70 +/- 5 vs 4 +/- 0.5 cpm/micrograms protein/h at 30 and 60 days, respectively) and in percentages of cell nuclei incorporating bromodeoxyuridine (BrDU) (from 46 +/- 6% to less than 1%). Replating at 10(4) cells/cm2 yielded secondary cultures which synthesized DNA actively. Forty-eight hours of serum deprivation at 2-3 days from subculturing, followed by addition of 10% serum (time 0), resulted in a return to quiescence which persisted until 12 h (G0 + G1). By 20 h (S phase), there were abrupt increases in DNA synthesis (5-fold) and in BrDU-labeled nuclei (from 19 +/- 2 to 76 +/- 8%) and the percentage of glial fibrillary acidic protein (GFAP)-positive cells declined to 14 +/- 2%. Three days later, GFAP-positive cells numbered around 80%. Cell cycling after prolonged quiescence, in a manner similar to that in early astroglial cultures, required a non-sterol derivative of mevalonate in late G1. These data confirm that astroglia in primary cultures, like their counterparts in vivo, have a flexible capacity to enter and depart from quiescence, and most importantly, provide a system for examining regulation of this process.

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PubMed1991

Isoprenoids and astroglial cell cycling: diminished mevalonate availability and inhibition of dolichol-linked glycoprotein synthesis arrest cycling through distinct mechanisms.

Primary astroglial cultures were used to compare the relationships to cell cycling of dolichol-linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift-up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 +/- 10%; DNA synthetic rates (thymidine incorporation) increased 5-fold. S phase transition was prevented by addition at 10-12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin-treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle-specific roles: dolichol-linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.

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PubMed1991

Cell cycling of astrocytes and their precursors in primary cultures: a mevalonate requirement identified in late G1, but before the G1/S transition, involves polypeptides.

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/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/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 G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.

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