Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures

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Abstract
The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15–30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5, 6 -3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37°C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr. The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4–10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied. Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures. The addition of EGF to DMEM + BSA significantly increased cell number, while addition of insulin had no effect. The highest values for cell number were observed when insulin and transferrin were added together with EGF. Moreover, EGF treatment in the presence of serum significantly increased the incorporation of labeled amino acids into vimentin, glial fibrillary acidic protein, and actin.

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