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

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 degrees 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.

Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (cx) 43 expression and function in astroglial cells from distinct brain regions

“…This notion is supported further by the fact that astroglial cells co-treated with FGF-2 and the antimitogen TGF-␤3 (Flanders et al, 1993) were entirely growth arrested without any change in the response to FGF-2 in terms of uncoupling. Finally, EGF, an established mitogen for astroglial cells (Avola et al, 1988), failed to reduce coupling, despite its promoting effect on cell proliferation. This adds further to the evidence that cell proliferation and coupling can be independently regulated in cultured astroglial cells by multifunctional cytokines, e.g., FGF-2.…”

Effects of Interleukin-1 and Tumor Necrosis Factor-α on Astrocytes, Microglia, Oligodendrocytes, and Glial Precursors in vitro

“…In the absence of IL1 and TNFa, no IL6 and PDGF are likely to be present. EGF, although not produced by astrocytes, is an astroglial proliferation and differentiation factor [8,9,43] and can act as such in synergy with 1GF-I. It may exist in serum in low levels.…”

“…In addition, the best response in that study was obtained in chemically defined medium, containing 5 pg/ml bovine insulin [33]. Insulin and IGFs can, in addition to acting as direct growth promoters, augment proliferation of cells in response to other factors such as EGF by acting as progression fac tors [8,34,35]. We used nanomole per liter concentra tions of TNFa because these are the levels produced by glia in vitro, levels which are biologically active.…”

“…The morpho logical changes in reactive astrocytes have been described as differentiation, in which the flat, polygonal or epithe lioid cells convert to stellate cells with slender un branched processes [2,6,7], Gliosis in vivo is visualized as an increased intensity in immunostaining of the astro cytic intermediate filament glial fibrillary acid protein (GFAP). Similar processes can be induced in astrocytes in vitro in response to a number of growth factors includ ing epidermal growth factor (EGF), and fibroblast growth factor (FGF) [8][9][10]. There is some question as to whether gliosis interferes with axonal and myelin repair after damage or destruction has occurred [2,5,6].…”

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

“…Previous works have shown that EGF can stimulate the proliferation of primary astrocyte cultures obtained from rat cerebral hemispheres (Avola et al 1988;1993), and it strongly affects the morphology of astrocytes and induces upregulation of the glutamine synthase and S-100 (Avola et al 1988;1993). In the present study, the addition of EGF to this modified chemically defined medium significantly improved the proliferative capability of cultured astrocytes.…”