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A M Otto

Publications and source records attributed to A M Otto.

At least 37 records · Page 2Linked to original sources
PubMed1984

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.

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PubMed1983

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.

Animals↗
PubMed1982

The effect of serum, EGF, PGF2 alpha and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis.

To test the connection between S6 phosphorylation and the activation of protein and DNA synthesis, we compared the effects of serum, epidermal growth factor (EGF), prostaglandin F2 alpha (PGF2 alpha) and insulin (which is not mitogenic in these cells). Increasing concentrations of serum or EGF produced roughly parallel effects on all three processes, though the maximum response elicited by EGF (10(-9) M) was only a portion of that caused by saturating levels of serum (7.5% to 10%). PGF2 alpha (8.5 x 10(-7) M) alone acted similarly to EGF (10(-9) M) and with EGF produced a synergistic effect on all three processes. Insulin (10(-9) M) alone stimulated both S6 phosphorylation and protein synthesis to approximately the same level as EGF or PGF2 alpha, but had no effect on initiation of DNA synthesis. Thus neither stimulation of S6 phosphorylation nor activation of protein synthesis is sufficient for initiation of DNA synthesis. The requirement for S6 phosphorylation could not be dissociated from the activation of protein synthesis. Ribosomes containing the most highly phosphorylated forms of S6 appear to have a selective advantage in entering polysomes.

Animals↗
PubMed1982

Prostaglandins E1 and E2 interact with prostaglandin F2alpha to regulate initiation of DNA replication and cell division in swiss 3T3 cells.

Prostaglandin (PG) E1 or E2 added at 2-1,000 ng/ml to quiescent cultures of Swiss 3T3 cells synergistically enhanced the rate of initiation of DNA replication stimulated by PGF2 alpha alone or with insulin. Neither PGD2 nor PGF1 alpha had any effect with PGF2 alpha. An increase in the rate of entry into S phase also occurred when PGE1 or PGE2 was added 8 or 15 hr after addition of PGF2 alpha. However, adding PGE1 and PGE2 together with PGF2 alpha did not further enhance the synergistic effect observed with PGE1 or PGE2 separately. The synergistic effect was also observed in stimulation of 2-deoxyglucose uptake but not in early changes of intracellular levels of cAMP. These results may be relevant in understanding the control of fibroblastic proliferation in wound healing and may provide an alternative mechanism for oncogenic transformation.

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PubMed1981

Glucocorticoids inhibit the stimulatory effect of epidermal growth factor on the initiation of DNA synthesis.

Confluent, quiescent Swiss 3T3 cells in culture can be stimulated to initiate DNA synthesis and divide by addition of growth factors to the culture medium. Here we show that hydrocortisone and other steroids which have glucocorticoid activity inhibit the stimulation of these cells by epidermal growth factor (EGF) in contrast to their reported enhancement of stimulation by fibroblast growth factor (FGF). Binding studies using [3H]-triamcinolone acetonide show that Swiss 3T3 cells contain a single class of glucocorticoid receptor of uniform affinity (KD = 2.0 nM), and about 34,000 receptor sites per cell. Those steroids which displace bound [3H]-triamcinolone acetonide are also effective in inhibiting the stimulation of DNA synthesis by EGF in the presence or absence of insulin, and the concentration of triamcinolone acetonide required for one-half maximal biological effect is in the same range as the KD. A similar concentration is required for one-half maximal enhancement of the effect of FGF. These results suggest that both the inhibitory and stimulatory effects of glucocorticoids may be mediated via these receptors, the different effects thus being due to differences in the intracellular events triggered by each growth factor.

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PubMed1981

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells--interactions with hormones and growth factors.

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14-15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4-6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases K, but after a delay of 4-6 hours. In contrast, both hydrocortisone and prostaglandin E1 (PGE1) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F2 alpha), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF2 alpha, nevertheless, act through different regulatory events.

Animals↗
PubMed1981

Two growth factors and two hormones regulate initiation of DNA synthesis in cultured mouse cells through different pathways of events.

Epidermal growth factor (EGF) as well as prostaglandin F2 alpha (PGF2 alpha), when added to quiescent, confluent Swiss 3T3 cells, stimulate the initiation of DNA synthesis, which occurs with apparent first-order kinetics after a lag phase of 14-15 hr. These two growth factors appear to stimulate similar events; insulin enhances and hydrocortisone can inhibit the stimulatory effect of either. Here we show that the addition of EGF and PGF2 alpha together, however, results in a synergistic effect seen at the end of the lag phase, but only when EGF and PGF2 alpha are added within 6 hr of each other. Addition of one growth factor 10 or 15 hr after the other delayed the synergy for 15 hr after the addition of the second growth factor. Insulin further increased the rate of entry into the s phage stimulated by EGF and PGF2 alpha together, whereas hydrocortisone inhibited the stimulatory effect observed with either EGF or PGF2 alpha alone. These results suggest that, in spite of the common events responsible for the interactions with the two hormones, EGF and PGF2 alpha must have differences in their sequences of events that initiate DNA synthesis.

Animals↗
PubMed1981

Microtubule-disrupting agents affect two different events regulating the initiation of DNA synthesis in Swiss 3T3 cells.

The stimulatory effect of epidermal growth factor, alone or with insulin, on the rate of initiation of DNA synthesis in Swiss 3T3 cells can be synergistically enhanced by the addition of either Colcemid or colchicine at 1 microM. However, both Colcemid and colchicine can exert the synergistic effect only when added earlier than 8 hr of the prereplicative period (lag phase). Removal of Colcemid (which allows for rapid reassembly of microtubules) earlier than 10 hr of the lag phase results in a loss of the synergistic effect. This suggests that microtubules must remain disrupted for longer times to accomplish some putative event(s) necessary for increasing the rate of initiation of DNA synthesis. Preincubation of quiescent cells with either Colcemid or colchicine for 8 hr prior to adding epidermal growth factor, alone or with insulin, shortens the lag phase by about 4 hr, irrespective of the resulting rate of initiation of DNA synthesis. These results suggest that the state of microtubules is affecting independently at least two different events involved in regulating time initiation of DNA synthesis.

Animals↗
PubMed1980

Cell cycle dependent rate of labelling of cellular and secreted glycosaminoglycans in mouse embryonic fibroblasts.

Cultures of embryonic fibroblasts from Balb/c or CBA/J mice were given 12-h pulses of 14C-galactose, or were double-labelled with 3H-galactose and 35H-sulfate. The time course of the rates of labelling of glycosaminoglycans--galactose label was found in the uronic acid moiety--was studied in synchronously and asynchronously growing cultures. Partial synchrony was achieved by trypsinising quiescent, confluent cells and subsequent transfer of cells to new cultures with fresh medium. Synchrony was monitored by measurement of thymidine uptake in parallel cultures. The distribution of label in the hyaluronic acid, chondroitin sulfate, and heparan sulfate fractions from cells and culture media was determined at each time point. Peaks of DNA synthesis were accompanied by or followed 12 h later by a maximal rate of labelling with galactose of secreted glycosaminoglycans, and with the exception of hyaluronic acid--also of cellular glycosaminoglycans. The rate of labelling with galactose of glycosphingolipids in parallel cultures followed a different time course. In double-label experiments the rates of labelling of glycosaminoglycan sulfates with 3H-galactose and 35S-sulfate did not go parallel. In older, quiescent cultures the labelling rate with galactose decreased while the sulfation rate increased. It is discussed that the labelling rate with galactose is indicative of the biosynthetic rate of the glycosaminoglycans. The conclusion is reached that glycosaminoglycans are preferentially synthesized and secreted after the S phase of the cell cycle.

Animals↗
PubMed1980

Dissociation of uridine and (86Rb+) uptake from stimulatioin of DNA synthesis in Swiss 3T3 cells.

The importance of the stimulation of uridine and (86Rb+) uptake to the stimulation of DNA synthesis was investigated using three defined growth factors, EGF, FGF, and PGF2 alpha, and three hormones, hydrocortisone, insulin and PGE1, which do not stimulate proliferation of Swiss 3T3 cells but modify the response to these growth factors. Uridine uptake is stimulated by insulin, but not by PGF2 alpha, indicating that its activation is neither sufficient nor necessary for stimulation of cell proliferation. (86Rb+) uptake was stimulated by each growth factor tested, but also by insulin and PGE1. Modifying effects of insulin, hydrocortisone or PGE1 in combination with growth factors on the level of DNA synthesis were not reflected in changes in stimulation of these uptake systems. We conclude that these events are regulated separately and are not tightly coupled to the initiation of DNA synthesis.

Animals↗
PubMed1979

Cytoskeleton-disrupting drugs enhance effect of growth factors and hormones on initiation of DNA synthesis.

Addition of growth factors, such as prostaglandin F2 alpha or fibroblastic growth factor, to quiescent Swiss mouse 3T3 cells resulted in an abrupt increase in the rate of initiation of DNA synthesis after a lag phase of 13-15 hr. This increase could be quantified by a rate constant k. Addition of colchicine, Colcemid, or vinblastine had a synergistic effect on the initiation of DNA synthesis triggered by PGF2 alpha or FGF by increasing the value of k. These drugs alone had no effect. Colchicine had a synergistic effect only if added within 8 hr of the PGF2 alpha or FGF addition. Also, colchicine exerted its full effect when it was present only for the first 5 hr with either growth factor. These results suggest that an intact cytoskeleton is not required for the initiation of DNA synthesis. Furthermore, cytoskeleton-disrupting drugs enhance the stimulatory effect of the growth factors.

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PubMedDate not supplied

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.

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