The role of endogenous growth inhibitors in the regulation of cell proliferation is unclear. Although there are numerous studies on the stimulatory effect of peptide hormones such as insulin, the somatomedins and epidermal growth factor (EGF) on cell proliferation, little is known about the existence of hormones that might exert an antiproliferative effect on cells. Somatostatin (SS), a cyclic tetradecapeptide hormone that is widely distributed in the body, exerts an inhibitory effect on numerous cellular processes. We observed that SS at concentrations of 10(-8) - 10(13M), inhibited EGF-induced centrosomal separation, which recent evidence suggests is necessary for DNA synthesis in response to EGF. This SS effect was associated with inhibition of DNA synthesis and cell replication induced by EGF in gerbil fibroma and HeLa cells. Inhibition of centrosomal separation and the consequent antiproliferative effect of SS may represent a biologically significant action of this ubiquitous hormone.

Using a rabbit antibody to MAP1 to stain centrosomes we have studied the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa cells. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and >70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.05 ng/ml) and 0.1 to 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine, and W-7, cytochalasins B and D, and taxol, and is stimulated or enhanced by A23187, colchicine, and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. Our hypothesis based upon these results is that EGF, by raising the free calcium level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.Epidermal growth factor (EGF) stimulates proliferation of a wide variety of cultured cells. After binding to specific cell membrane receptors the hormone sets in motion a chain of events which lead eventually to cell replication (8,12). A minimum interval of 12-15 h exists between EGF binding and the onset of DNA synthesis (2, 3, 10, 27, 31) which suggests that a major reorganization of the biochemical and structural machinery of the cell must occur. In fact, numerous biochemical and morphological changes have been described in response to EGF (for reviews see references 8 and 12), including increases in glycolysis (19), ion (48), amino acid (28), and hexose transport (4), membrane ruffling (7, 14), hormone and receptor uptake (9, 38), and protein tyrosine phosphorylation (11, 29). However, despite the accumulation of a great deal of information about what happens after EGF binding we are still far from understanding the complex program through which a cell prepares itself for a cycle of DNA replication and division in response to EGF or other mitogens.It has been shown that colchicine induces DNA synthesis in quiescent cells (17,52) and enhances the mitogenic effect of EGF and other growth factors (17,22,37,42,43,52). Furthermore, taxol, a drug which prevents microtubule disassembly, has recently been shown to inhibit EGF and thrombin-stimulated DNA synthesis in mouse embryo cells (16). These studies suggest that microtubule disassembly or reorganization is involved in the regulation of cell replication.We recently reported that EGF stimulates centrosomal separation in 3T3 and H...

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

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...

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