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

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 G0/G1 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.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

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.

Tumor promoters stimulate hyperplasia of microtubule organizing center and inhibit DNA synthesis in cultured cells.

Chemical tumor promoters induce significant morphologic changes in several cultured cell models. In this article we describe a new effect of two potent, chemically different tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and dihydroteleocidin B (DHTB) on cultured human HeLa and melanoma cells. Using immunofluorescence microscopy, we observed that TPA and DHTB induced a dramatic increase in the size (greater than or equal to 3X normal diameter) of the centrosome, a microtubule-organizing center, within 24 h of incubation. In HeLa cells the effect was serum- and dose-dependent, was observed in 76-92% of cells within 72 h of incubation, and was associated with an increase in cytoplasm-nucleus ratio and proliferation of microtubules from the centrosome. The tumor promoters inhibited serum-induced DNA synthesis in both cell lines. Electron microscopy revealed the presence of clumps of microcentriole bodies or fragments adjacent to the intact centriole.

Insulin and multiplication-stimulating activity induce a very rapid centrosomal orientation response to wounding in endothelial cell monolayers.

Using serum-deprived monolayers of bovine aortic endothelial cells that were disrupted experimentally with a linear wound, we observed, by immunofluorescence microscopy, centrosomal perinuclear movement that is associated closely with cell orientation and locomotion. In the presence of the growth factors fetal bovine serum, insulin, or multiplication-stimulating activity (MSA), the centrosome rapidly (within 10 s) translocated and positioned itself between the nucleus and wound track in greater than or equal to 70% of cells bordering the wound. Random centrosomal orientation was observed in control border cells (50%). This growth factor-stimulated centrosomal orientation response to wounding depended on growth factor concentration, high-energy phosphates, functional microtubules and microfilaments, and calcium-calmodulin interaction. In the absence of fetal bovine serum, insulin, or MSA, the border cells of the wounded endothelial cell monolayer exhibited a significant centrosomal orientation response 5-6 h after wounding.

Glyburide modulates insulin-mediated locomotor response of confluent cell cultures to wounding.

We determined whether the sulfonylurea glyburide could modify the insulin-induced locomotor response to wounding of bovine aortic, pulmonary artery endothelial, and gerbil fibroma cells. Serum-deprived cells were preincubated in medium containing glyburide (10(-4) M) or diluent, then exposed to insulin (10(-6) to 10(-12) M) at the time of wounding, after which the centrosomal-orientation and migratory responses of the cells bordering the experimental wound were monitored by immunofluorescence microscopy and time-lapse cinemicrophotography, respectively. Insulin (10(-6) M) stimulated the centrosomal-orientation and locomotor responses, resulting in the narrowing of the linear wound. Glyburide enhanced the effect of insulin on both parameters of the locomotor response and also sensitized the cells to a previously ineffective concentration of insulin (10(-12) M). The action of glyburide depended on a preincubation period of greater than or equal to 60 min and was blocked by cycloheximide and actinomycin D. The relevance of this novel action of glyburide to the impaired healing process observed in diabetic patients requires further study.

Somatostatin inhibits rapid centrosomal separation and cell proliferation induced by epidermal growth factor.

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.

The effects of hyperglycemia on the directed migration of wounded endothelial cell monolayers.

To determine if cellular motility is affected by abnormal concentrations of ambient glucose, we utilized an endothelial cell monolayer model of wounding to observe the effect of hyperglycemia on the serum-mediated polarization and migration responses of cells at the edge of the wound. We found that hyperglycemia inhibited the rapid polarization response and the consequent cellular migration induced by 20% fetal bovine serum. The inhibitory effect of glucose was significant at a concentration of 11 mmol/L and became more pronounced at higher glucose concentrations. The effect of hyperglycemia on the two components of cell locomotion differed from the inhibitory action of mannitol, another hyperosmolar agent, in two ways (1) the glucose-induced inhibition persisted in cells reincubated in medium containing physiologic glucose (5.5 mmol/L); and (2) the inhibitory effect of glucose was blocked in cells preincubated in medium containing either the sulfonylurea glyburide, or D-myo-inositol. These observations may be relevant to the pathogenesis and management of accelerated atherosclerosis associated with the diabetic state.

The cysteine protease inhibitor, E-64, stimulates the polarization and locomotor responses of endothelial cells to wounding.

To clarify the role of proteases and protease inhibitors in the initiation and execution of endothelial cell movement, we observed the effect of several protease inhibitors on the polarization and locomotor responses of an endothelial cell monolayer subjected to experimental wounding. We found that the thiol protease inhibitor, E-64 (L-transepoxysuccinyl-leucylamido-[4-guanidino]butane) stimulated both cellular processes. The stimulatory effect of E-64 on the polarization response of cells to wounding required a preincubation period of at least 1 hr, calcium-calmodulin interaction, protein kinase C activation, and was blocked by cyclic AMP analogs. The chemokinetic action of E-64 appears to be unique among the protease inhibitors tested and may represent a novel role for this cysteine protease inhibitor or its endogenous counterpart in the modulation of cell locomotion.

Co-ordinate control of centrosomal separation and DNA synthesis by growth regulators.

We have tested the effects of various mitogens and growth inhibitors on centrosomal separation (CS) in serum-deprived HeLa, gerbil fibroma (GF) and A431 cells. All of the agents which were mitogenic in a given cell type also stimulated CS. No agent was found which stimulated CS but failed to stimulate DNA synthesis. Inhibitors of DNA synthesis, including somatostatin, hydrocortisone, 8-bromo-cAMP, and epidermal growth factor (EGF) in A431 cells, also inhibited CS in response to mitogens. In GF cells (blocked at the G1/S interface with hydroxyurea) centrosomal re-association and the decay in commitment to DNA synthesis upon serum withdrawal occurred with a similar t1/2 (8.8 h). These results demonstrate that CS and DNA synthesis are co-ordinately regulated by a variety of stimulators and inhibitors of cell proliferation. Separation of the centrosomes, or an underlying event with which it is tightly coupled, may represent the point of cellular commitment to enter S phase.

Somatostatin has an antiproliferative effect on concanavalin A-activated rat thymocytes.

Somatostatin, a cyclic tetradecapeptide which is widely distributed in different tissues of the body, exerts an inhibitory effect on numerous cellular processes. It has been observed recently that somatostatin and its analogs were antimitogenic in several established cultured cell lines and in rat tumors. To determine whether this ubiquitous peptide had an antiproliferative effect on a primary culture of rat thymocytes, we observed its effect on the separation of the centrosome (a cell maker of the G1 to S traverse) and DNA synthesis of rat thymocytes activated by concanavalin A. Somatostatin inhibited both indices of proliferation in rat thymocytes at a concentration of 10(-8)M. This observation suggests that somatostatin, which has been localized in the thymus, may play a regulatory role in the growth and development of the cells found in the thymus gland, and may affect thymocyte function in disease states characterized by elevated circulating concentrations of somatostatin.

Localization of high molecular weight microtubule-associated proteins (MAP1 and MAP2) in a HeLa microtubule-organizing centre.

With a rabbit antiserum specific for high molecular weight microtubule-associated proteins (HMW/MAPs: MAP1 and MAP2), centrosomes in interphase and mitotic HeLa cells were visualized by indirect immunofluorescence. Pre-immune serum did not stain a similar organelle. Microtubules were observed to originate from the centrosomes, demonstrating their microtubule organizing (MTOC) function. The MTOC antigen(s) to which the anti-HMW/MAP antiserum reacted was resistant to Colcemid, Triton-X-100, Nonidet P-40 and Brij 35. Immunofluorescence localization of the MTOCs was blocked by pre-incubation of the antiserum with a MAP1--MAP2 preparation but not with tubulin, actin, or a 58 K intermediate filament protein.

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity.

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.