Epidermal growth factor induces rapid centrosomal separation in HeLa and 3T3 cells.

Abstract

Using indirect immunofluorescence, we have found that epidermal growth factor (EGF), at 100 ng/ml, induces centrosomal separation within 20 min in HeLa and 3T3 cells. The effect was evident both in unsynchronized cultures and in HeLa cells blocked in early S phase by hydroxyurea. EGF also induced centrosomal separation in quiescent 3T3 cells blocked in Go/G 1 by serum deprivation, indicating that DNA replication is not necessary for this effect . The mechanism of this rapid centrosomal separation and its role in the mitogenic effects of EGF remains to be determined .Epidermal growth factor (EGF) (6,045 mol wt) is probably the most extensively studied mammalian mitogenic peptide (6) . EGF was originally isolated from mouse submaxillary glands but it is clearly made by other tissues because submaxillary gland excision does not lower plasma levels of EGF (4) . In man, EGF is found in plasma, saliva, and urine but its precise physiological role is unknown (13, 37). Since a wide variety of cells of epithelial (19,33,39), fibroblastic (1, 2, 5, 21), and neuronal (45) origin are stimulated to proliferate in vitro in response to EGF, it seems that this peptide is likely to be an important regulator of cell division in vivo.As with all other peptide hormones studied thus far, the initial event in the action of EGF is binding to specific highaffinity receptors on the external surface of the plasma membrane (1, 5, 6, 21); 15-22 h later DNA synthesis ensues with subsequent cell replication (1,5,6,22) . Although it is clear that major changes in the organization and function ofthe cell must occur in response to EGF before cell division, the key events leading from membrane binding to cell division are unknown.Centrosomes are perinuclear structures which consist of a pair of centrioles surrounded by an electron-dense cloud of pericentriolar material (34,40). During interphase many cytoplasmic microtubules originate from this structure (14,17,29). During mitosis a centrosome is located at each spindle pole and serves as an anchoring site for microtubules that make up the spindle apparatus . The centriole cycle, which includes duplication, elongation, and polar migration is, in general, believed to be tightly coupled to the cell cycle but the precise relationship between centriole and cell-cycle events remains to be elucidated. For example, while centriole formation can proceed in the absence of DNA synthesis (26, 32) it does require the presence of the nucleus (26). Whether centrosomal behavior influences nuclear events is an open question.Using a specific antiserum to a high molecular weight microtubule-associated protein (MAP,), which stains centrosomes THE JOURNAL OF CELL BIOLOGY " VOLUME 93 MAY 1982 507-511 © The Rockefeller University Press " 0021-9525/82/05/0507/06 $1 .00 in a variety ofcells, we have found that, after addition of EGF, the centrosome splits . The paired daughter centrosomes maintain close contact with the nucleus but migrate away from one another along the nuclear surface . The rapidity ofthe re...

Mammalian RanBP1 regulates centrosome cohesion during mitosis

“…1B: cells with one or two paired dots (corresponding to one or two centrosomes, respectively) were taken as normal, whereas cells with more than two pairs of dots or with scattered dots were assumed to reflect overduplication and abnormal splitting of centrosomes, respectively, and considered to be abnormal. Serum restimulation of quiescent cells induces per se a high frequency of centrosome splitting in vector-transfected cells (Table 1), in line with previous reports (Sherline and Mascardo, 1982;Schliwa et al, 1982;Schliwa et al, 1983). RanBP1 overexpression had no additional effect on serum-induced centrosomal abnormalities in interphase; however a significant increase was recorded in RanBP1-overexpressing mitotic cells compared to control cultures (Table 1).…”

Protein kinases in control of the centrosome cycle

“…Clearly, being active in G2 and a putative regulator of C-Nap1, Nek2 is an ideal candidate kinase to trigger this event. However, it should be pointed out that serum stimulation and drug treatment can also induce transient splitting in interphase cells at a time when Nek2 is not supposed to be active [33,34]. Indeed, we found recently that overexpression of certain other active kinases can also trigger centrosome splitting, albeit to di¡erent Fig.…”

Catecholamines are mitogenic in 3T3 and bovine aortic endothelial cells.

“…We recently found that stimulation of DNA synthesis by epidermal growth factor was associated with rapid centrosomal separation in a variety of cell types (1)(2)(3). In the course of investigating the effects of other growth factors on centrosomal separation we found that epinephrine and norepinephrine also increased centrosomal separation, which suggests that catecholamines might be mitogenic as well.…”