Leukaemia inhibitory factor induces mitogenesis in Swiss 3T3 cells and selective enhancement via a variety of signalling events.

Leukaemia inhibitory factor (LIF) stimulates cellular DNA synthesis in confluent quiescent Swiss 3T3 cells. Insulin and prostaglandin E1 (PGE1), which fail to stimulate DNA synthesis alone, potentiate this effect. Prostaglandin F2alpha (PGF2alpha), which is mitogenic in these cells, enhances the effect of LIF on DNA synthesis. TGFbeta1 increases the effect of PGF2alpha but not that of LIF. R-59022, a diacylglycerol kinase inhibitor which increases protein kinase C (PKC) activity, enhances only the PGF2alpha response. 13-Tetradecanoyl-12-phorbolacetate-mediated PKC depletion prevents the action of PGF2alpha but not that of LIF, nor the PGF2alpha potentiation of LIF-stimulated DNA synthesis. 1-Oleoyl-2acetylglycerol, a PKC and tyrosine kinase (TK) activator which mimics some of the PGF2alpha effects, enhances only LIF-induced DNA synthesis in cells possessing intact PKC activity. These results suggest that stimulation of DNA synthesis by LIF, as well as its enhancement by PGF2alpha, may occur via a signalling pathway independent of PKC activation.

A mitogenic and hormonal signalling network regulate mammalian cell division commitment time.

A basic property of mammalian cells is to retain the mitogenically induced "commitment" to undergo DNA replication even in the absence of stimuli. Recent findings on PGF2 alpha and hormone-induced Swiss 3T3 cell multiplication, reveal that this crucial cell cycle event can be regulated by several signalling mechanisms.

Prostaglandin F2 alpha (PGF2 alpha) triggers protein kinase C (PKC) and tyrosine kinase activity in cultured mammalian cells.

Prostaglandin F2 alpha (PGF2 alpha) added to confluent resting Swiss 3T3 cells triggers tyrosine kinase (PTK) activation characterized by the phosphorylation of a set of 75, 86, 110 and 140 kD proteins. PGF2 alpha induces this event independently of PKC activation. However, both PKC and PTK activities appear to act concertedly to cause mitogenesis. Here we discuss their relevance in the control of mammalian cell division.

Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture.

Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2 alpha in Swiss mouse 3T3 cells.

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)- or prostaglandin F2 alpha (PGF2 alpha)-induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2-80 microM concentration range, with both mitogens attaining 50% inhibition at 7.5 microM. LOV exerted its effect within 0-8 h following mitogenic induction. Mevanolactone (10-80 microM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF2 alpha response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF2 alpha-stimulated cells, LOV did not inhibit [3H]leucine or [3H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N' glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N' glycosylation process. These findings strongly suggest that either EGF or PGF2 alpha stimulations generate early cell cycle signals which induce mevalonate formation, N' glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed.

Transforming growth factor beta 1, insulin and prostaglandin E1 enhance prostaglandin F2 alpha mitogenic action in Swiss 3T3 cells via separate events.

Transforming growth factor beta 1 (TGF beta 1) had no mitogenic effect in Swiss 3T3 cells, but could increase prostaglandin F2 alpha (PGF2 alpha)-induced DNA synthesis. Insulin, but not prostaglandin E1 (PGE1), further enhanced PGF2 alpha action at low TGF beta 1 concentrations. TGF beta 1 also acted concertedly with the protein kinase C (PKC) activator 1-oleoyl-2-acetylglycerol to induce mitogenesis. Thus, it appears that TGF beta 1 and insulin act via separate signals, while TGF beta 1 and PGE1 might share a common pathway not involving TGF beta 1-mediated prostaglandin synthesis. These results suggest that TGF beta 1 might elicit various signalling mechanisms to enhance PGF2 alpha-triggered events.

Early cell cycle diacylglycerol (DAG) content and protein kinase C (PKC) activity enhancement potentiates prostaglandin F2 alpha (PGF2 alpha) induced mitogenesis in Swiss 3T3 cells.

R59022, a diacylglycerol kinase inhibitor, enhances the prostaglandin F2 alpha (PGF2 alpha)-induced diacylglycerol (DAG) synthesis in Swiss 3T3 cells. It also potentiates the PGF2 alpha-mediated protein kinase C (PKC)-dependent 80 kDa protein (80K) phosphorylation and initiation of DNA replication. R59022 enhances the PGF2 alpha mitogenic response by increasing the rate of entry into the S phase. Insulin does not cause 80K phosphorylation, and does not enhance its induction but it potentiates the PGF2 alpha mitogenic response. These results suggest that mitogenically triggered fluctuations in DAG content and PKC activity play a pivotal role in controlling the PGF2 alpha-induced DNA synthesis while insulin acts via a different mechanism.

Prostaglandin F2 alpha decreases the affinity of epidermal growth factor receptors in Swiss mouse 3T3 cells via protein kinase C activation.

Prostaglandin F2 alpha (PGF2 alpha) selectively decreases the binding of 125I-labelled epidermal growth factor ([125I]EGF) to intact Swiss 3T3 cells. Scatchard analysis reveals that PGF2 alpha decreases the number of high-affinity EGF binding sites without changing the total number of receptors. Prostaglandins E1 (PGE1), E2 (PGE2) or F2 beta (PGF2 beta) do not alter the EGF binding to these cells and do not enhance the PGF2 alpha effect. R-59022 and R-59949, two diacylglycerol kinase inhibitors, enhance the inhibitory effect of PGF2 alpha, whereas down-modulation of protein kinase C (PKC) abolishes the effect. These results indicate that PGF2 alpha decreases EGF binding in Swiss 3T3 cells via PKC activation.

Stimulation of protein kinase C (PKC) activity in resting Swiss 3T3 cells by prostaglandin F2 alpha.

Prostaglandin F2 alpha (PGF2 alpha), a mitogen for resting Swiss 3T3 cells, rapidly stimulates phosphorylation of an 80 kDa protein (80 K). 1-Oleoyl-2-acetylglycerol (OAG) and 12-O-tetradecanoyl phorbol-13-acetate (TPA) both protein kinase C (PKC) activators, also elicit 80 K phosphorylation. In contrast PGE1, PGE2 or PGF2 beta, which are non-mitogenic in these cells, had little or no action on this event. However PGE1 and PGE2 potentiate the PGF2 alpha proliferative effect but do not enhance its action on 80 K phosphorylation. These results suggest that PGF2 alpha mitogenic induction involves PKC signalling pathway activation while its enhancement by PGE1 or PGE2 occurs through a different mechanism(s).

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2 alpha in Swiss mouse 3T3 cells.

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F2 alpha alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF2 alpha stimulation. In contrast tunicamycin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF2 alpha.

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase alpha activity.

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.

Increase of epidermal growth factor-stimulated cell-cycle progression and induction of thermotolerance by heat shock: temperature and time relationship.

When quiescent confluent cultures were incubated at increased temperature and then incubated at 37 degrees C prior to a second increase of temperature (46 degrees C) it appeared that heat-induced morphological alteration and ability to proliferate could be influenced by the previous thermal history of the cells. Incubations for 20 min in a temperature range of 41-46 degrees C caused cells to develop thermo-tolerance within 3 h of incubation at 37 degrees C. Confluent quiescent Swiss mouse 3T3 cells were incubated at 41.8, 43.7 or 45.6 degrees C and then reincubated at 37 degrees C to determine the effects of heat shock on the mitogenic effects of epidermal growth factor (EGF). Preincubation at 43.7 degrees C or 45.6 degrees C enhanced stimulation of G1-S progression by EGF. Preincubation at 43.7 degrees C markedly increased the rate at which cells enter the S phase without changing the length of the lag phase. A comparison of the duration of incubation at 43.7 degrees C for potentiation of EGF-induced DNA synthesis and that for induction of thermotolerance showed that a similar time interval for induction of effect could be implied.

Hyperthermia can enhance the initiation of DNA synthesis stimulated by growth factors in swiss mouse 3T3 cells.

Confluent quiescent Swiss mouse 3T3 cells can be stimulated to initiate DNA synthesis and to divide by epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha), two mitogens of unrelated structure. Heat treatment at 46 degrees C for up to 20 min of confluent quiescent cells, which has no mitogenic effect, can enhance the stimulatory effect of suboptimal concentrations of EGF or PGF2 alpha on the initiation of DNA synthesis. Furthermore, insulin, which is not mitogenic in these cells, enhances the effect of these mitogens, but this effect is not further enhanced by heat treatment. Likewise the combination of EGF and PGF2 alpha is synergistic on DNA synthesis, and this effect is also not enhanced by the heat treatment. Incubation at 46 degrees C for longer than 20 min was inhibitory in all cases. These results suggest that heat treatment induces events which affect the regulation of the initiation of DNA synthesis in a manner depending on the duration of the heat treatment and the stimulation of the cells.

Effect of serum and growth factors on heat sensitivity in Swiss mouse 3T3 cells.

Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46 degrees C for 20 min by changing their flat, well-extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37 degrees C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha) markedly increased the heat resistance to a 46 degrees C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2-3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF2 alpha in these cells) was added alone or in combination with either EGF or PGF2 alpha, it has no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2-4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46 degrees C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells.

Prostaglandin F2 alpha stimulates phosphatidylinositol turnover and increases the cellular content of 1,2-diacylglycerol in confluent resting Swiss 3T3 cells.

Prostaglandin F2 alpha (PGF2 alpha); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2 alpha when compared with PGE1, PGE2, and PGF2 beta. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2 alpha to enhance DNA synthesis, does not affect the ability of PGF2 alpha, to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2 alpha, also causes a small increase in the cellular content of 1,2-diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2-diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2 alpha differs in its action from EGF.

The stimulation of the initiation of DNA synthesis and cell division in Swiss mouse 3T3 cells by prostaglandin F2 alpha requires specific functional groups in the molecule.

Among a number of prostaglandins, PGF2 alpha had the highest specific activity for stimulating the initiation of DNA synthesis in confluent resting Swiss 3T3 cells. At a saturating concentration of 8.5 X 10(-7) M, PGF2 alpha stimulated 21% of the cells to incorporate [ methyl-3H ]thymidine within 28 h. To elicit similar effects, prostaglandins F1 alpha, E1, E2, and D2 were required in 10-fold higher concentrations. Prostaglandins A1, A2, B1, and prostacyclin had no mitogenic activity. Insulin at 10(-8) M enhanced the stimulatory effect of PGF2 alpha and also of prostaglandins F1 alpha, E1, E2, and D2 by increasing the fraction of labeled nuclei. Methyl derivatives of PGF2 alpha were as effective as PGF2 alpha. Epimerization of the hydroxyl group at C-9 abolished the activity of the molecule. In contrast, upon epimerization at C-11 and C-15, some mitogenic activity was retained. In the presence of insulin, the latter molecules were as active as PGF2 alpha. Oxidation of the hydroxyl group at C-15 to a ketone abolished the mitogenic effect, while methyl ether formation led to only a slight loss of activity. Reduction of the delta 13 double bond also led only to a small reduction of activity. Similar differences in the activity of the various prostaglandins and analogues of PGF2 alpha were observed for 2-deoxyglucose uptake and increases in cell number. The relationships between structure and activity of prostaglandins suggest the existence of some specific receptor for PGF2 alpha to confer mitogenic response.