Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Effect of epidermal growth factor on synthesis of prostaglandins and cyclic AMP by embryonic palate mesenchymal cells

The influence of epidermal growth factor (EGF) on the ability of murine embryonic palate mesenchymal (MEPM) cells to be stimulated to synthesize cAMP and prostaglandins was investigated. Preincubation of MEPM cells with EGF enhanced, in a dose-dependent fashion, (1) the responsiveness of MEPM cells to prostaglandin E1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E2 and F2 alpha. Hormonal responsiveness of MEPM cells to EGF, prostaglandins and cAMP has been implicated as being involved in controlling various aspects of normal oro-facial development. We show here that EGF can potentiate hormonal responsiveness of these cells and thus allows consideration of EGF as a factor which may modulate hormonally regulated craniofacial growth and differentiation.     

Changes in the G0 state of WI-38 fibroblasts at different times after confluence

Some events in the prereplicative phase of WI-38 human diploid fibroblasts stimulated to proliferate are found to be a function of the length of time the cells have been quiescent. At 5 days after plating, when the cells first become confluent, the prereplicative phase upon stimulation by a nutritional change is relatively short, DNA synthesis begins at 8 h after stimulation, and there is no increase in chromatin template activity. At 9 days after plating the prereplicative phase of stimulated cells is lengthened to 14 h and there is an increase in chromatin template activity within 1 h of stimulation. Finally, in 18-day cells, the prereplicative phase is lengthened even further to 20 h, and there is a lag after stimulation before the increase in chromatin template activity. It is proposed that confluent WI-38 cells initially arrest in G 1, subsequently pass into G 0, and continue to go deeper into G 0 as they remain quiescent.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.     

Regulation of dog thyroid epithelial cell cycle by forskolin, an adenylate cyclase activator

Dog thyroid epithelial follicular cells in primary culture are quiescent in an insulin-supplemented serum-free medium. They are induced, after a 16- to 20-h prereplicative phase, to synthesize DNA upon stimulation by forskolin, a general adenylate cyclase activator that mimics all the effects of thyrotropin in these cells. The characteristics of adenylate cyclase activation by forskolin make this drug a convenient tool to enhance cellular cyclic AMP levels for well-defined periods of the cell cycle, allowing determination of which parts of the prereplicative phase are controlled by cyclic AMP. We observe that induction of DNA synthesis by forskolin requires its continuous presence for most of the prereplicative phase until a point that little precedes the initiation of DNA replication. Before this point, interruptions in forskolin presence as short as 2 h delay the onset of DNA synthesis, indicating a rapid regression of the cells to an earlier part of G1 from which they can be rescued by forskolin readdition. Similar delays in the onset of S phase are also induced by reversible protein synthesis inhibitions using pulses of cycloheximide. These data suggest that in dog thyrocytes elevated cyclic AMP levels stimulate the progression into G1 phase until a late commitment point before DNA synthesis. This progression depends on peculiarly labile cyclic AMP-stimulated events which might well be the induction by cyclic AMP of the synthesis of labile proteins.     

Comparative effects of TGFβ on proliferation of 7- and 14-day-old chick embryo fibroblasts and lack of involvement of the ODC/PA system in the TGFβ signaling pathway

The growth regulatory activity of transforming growth factor β (TGFβ) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of ³H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFβ treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFβ proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFβ on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFβ signaling. J. Cell. Physiol. 178:304–310, 1999. © 1999 Wiley-Liss, Inc.     

Cyclic AMP inhibits the release of prostaglandins and arachidonic acid from cultures of mouse embryo palate mesenchyme cells

Mouse embryo palate mesenchyme (MEPM) cells are able to synthesize and respond to prostaglandins. However, mechanisms that regulate their synthesis in these cells are not known. Cyclic adenosine 3',5' monophosphate (cAMP) has been implicated as being involved in differentiation of the palate, accumulates in MEPM cells in response to stimulation with selected prostaglandins, and has been found to modulate synthesis of prostaglandins by other cells and tissues. Therefore, we have investigated whether cAMP modulates synthesis of prostaglandins by MEPM mesenchyme cells and partially characterized the metabolic site at which such modulation occurs. We found that treatment of MEPM cells with various agents to stimulate a seven- to 100-fold increase in intracellular levels of cAMP inhibited release of various prostaglandins by at least 50%. Similarly, elevation of intracellular levels of cAMP inhibited release of radiolabeled arachidonic acid from membrane phospholipids by as much as 27%. The inhibitory effects of cAMP on release of prostaglandins from MEPM cells could be almost completely overcome by the addition of arachidonic acid to the culture medium. We interpret these data to mean that there is a regulatory cycle in MEPM cells in which intracellular levels of cAMP regulates synthesis of prostaglandins and prostaglandins regulate accumulation of cAMP and regulation of synthesis of prostaglandins by cAMP is predominantly through inhibition of a phospholipase.     

Cell cycle regulation by environmental pH

Purified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC-22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18-20-hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC-22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G₁ DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non-cell-cycle-phase-specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH-dependent restriction point in the G₁ phase of these tumour cells. The implications of these observations to tumour biology are discussed.     

Tyrosine protein kinase expression in long-term quiescent WI-38 cells following growth factor simulation

We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long-and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells “competent” to respond to the “progression” growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells. © 1995 Wiley-Liss, Inc.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Variation in the length of the lag phase following serum restimulation of mouse 3T3 cells

We have investigated the length of the lag phase (time taken for the first cells to enter S phase) and the kinetics of entry into DNA synthesis after serum restimulation of Swiss mouse 3T3 cell cultures that were allowed to become quiescent under different conditions. Cells were allowed to reach quiescence as a confluent monolayer in medium containing 10% (v/v) calf serum. Alternatively, when serum was reduced to 1% (v/v), cultures became quiescent at about 30% confluency and there was little cell to cell contact. The results show that the lag, or prereplicative phase becomes longer as the time spent in the quiescent state increases. This is the case in both confluent and non-confluent cultures. The rate of entry of cells into the S phase, however, remains the same under all conditions.     

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.     

Cyclic adenosine-3',5'-monophosphate alters hydrolysis of phospholipids by mouse embryo palate mesenchyme cells

The purpose of this study was to determine whether inhibition of release of arachidonic acid from mouse embryo palate mesenchyme (MEPM) cells in response to cAMP is due to a selected or generalized inhibition of hydrolysis of esterified pools of arachidonic acid. The calcium ionophore A23187 proved to be a useful probe of phospholipid hydrolases in MEPM cells, since it stimulated release of radiolabeled fatty acids from phospholipids of prelabeled MEPM cells as a function of the length of exposure, concentration, and concentration of Ca2+ in the medium. Elevation of intracellular levels of cAMP by treatment with (-) isoproterenol resulted in the inhibition of release of radiolabeled arachidonic acid in response to A23187. Analysis by quantitative gas-liquid chromatography revealed the source of the arachidonic acid released in response to the ionophore to be 1,2-diradyl-sn-glycero-3-phosphoethanolamine; elevation of intracellular levels of cAMP inhibited hydrolysis of this substrate, but may have stimulated hydrolysis of 1,2-diradyl-sn-glycero-3-phosphocholine. These findings permit the conclusions that 1) the ionophore stimulates activities of selected phospholipases A in MEPM cells and 2) cAMP modulates certain phospholipases A in MEPM cells in a specific manner.     

The stimulation of the initiation of DNA synthesis by fibroblast growth factor in swiss 3T3 cells: Interactions with hormones during the pre-replicative phase

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13–15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E₁ (PGE₁) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF. These results show that the stimulation of growth by FGF follows the same basic pattern as was previously shown with Prostaglandin F_2α (PGF_2α). However, since hydrocortisone inhibits stimulation by PGF_2α when added during the first 4 hr of the lag phase, there are clearly differences in some events stimulated by the two growth factors.