Enhanced DNA Synthesis Research Articles

Activation and proliferation signals in murine macrophages: Relationships among c‐fos and c‐myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis

Murine bone marrow-derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor-1 (CSF-1) and granulocyte-macrophage CSF (GM-CSF). These macrophages can also be "activated," but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl-methionyl-leucyl-phenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c-myc mRNA and c-fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.

Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12- O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serumtreated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.

Transforming growth factor‐β and γ‐interferon have dual effects on growth of peripheral glia

The influence of transforming growth factor-beta (TGF-beta) and gamma-interferon on DNA synthesis in Schwann cells and enteric glia in culture has been studied. TGF-beta stimulated the DNA synthesis of short-term (less than 2 weeks in culture) Schwann cells, whereas gamma-interferon was ineffective. The stimulatory effect of TGF-beta was additive to the stimulation of DNA synthesis due to axonal membrane fragments. In contrast to their effect on short-term Schwann cells, both TGF-beta and gamma-interferon inhibited DNA synthesis in enteric glial cells and in long-term (over 3 months in culture) Schwann cells. When short-term Schwann cells were stimulated to divide by axolemma or glial growth factor, gamma-interferon did not inhibit this enhanced DNA synthesis although it suppressed DNA synthesis induced by cAMP analogues. These results raise the possibility that TGF-beta and gamma-interferon might have a role in controlling glial proliferation during development and/or regeneration of the peripheral nervous system.

Further characterisation of ‘hepatotropin’, a high molecular weight hepatotrophic factor in rat serum

We report further characterisation of the hepatocyte growth factor ‘hepatotropin’ which is found in rat serum 24 h after partial hepatectomy. Hepatotropin enhances DNA synthesis in primary cultures of adult rat hepatocytes, and is of high molecular weight. Serum fractions were separated by gel filtration, heparin-sepharose affinity chromatography and ion-exchange chromatography. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) identified a band of apparent subunit relative molecular weight ( M r) 100 000 associated with biological activity, although purification to homogeneity has not been achieved. The activity is heat-labile, trypsin-sensitive, and stable to lyophilisation, but loses activity after dilution and reconcentration. In combination with known peptide hepatocyte growth modulators, hepatotropin acted synergistically with insulin and epidermal growth factor (EGF) but its action was not enhanced by glucagon. Studies on isolated rat hepatocyte membranes showed no evidence of enhanced phosphorylation of the EGF receptor by hepatotropin. The relationship of hepatotropin to previously described serum and platelet-derived growth factors is discussed.

Diet-induced reduction in serum lipoproteins stimulates cell proliferation in weanling rats

Male weanling Wistar rats were fed a 4% cholestyramine diet and used as a model to demonstrate that a reduction in serum low density lipoproteins stimulates de novo cholesterogenesis leading to DNA synthesis and cell proliferation. Feeding this diet resulted in a decrease in serum very low density lipoproteins and low density lipoproteins, an increase in high density lipoproteins and an increase in de novo cholesterogenesis in liver, thymus, spleen, pancreas, kidney and lung. DNA synthesis increased only in the thymus and spleen. Histological examination of spleen, thymus and lymph nodes showed an increased number of immature cells and enhanced mitotic activity. These results suggest that a marked reduction in serum low density lipoproteins stimulates de novo cholesterogenesis, leading to enhanced DNA synthesis and cell proliferation.

Lipoproteins May Provide Fatty Acids Necessary for Human Lymphocyte Proliferation by Both Low Density Lipoprotein Receptor-dependent and -independent Mechanisms

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptor-mediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (less than 10 micrograms cholesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (greater than or equal to 50 micrograms cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL3 enhanced responses of both normal and familial hypercholesterolemia lymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDL3. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptor-mediated and LDL receptor-independent interactions.

Parathyroid hormone-related protein modulates the effect of transforming growth factor-beta on deoxyribonucleic acid and collagen synthesis in fetal rat bone cells.

Proteins with biochemical function and sequence similarity to PTH are produced by many tumors associated with hypercalcemia and may have a role in pathological bone remodeling. Synthetic polypeptides comprising the amino-terminus of human PTH-related protein (PTH-rp) were examined for effects in intact fetal rat calvariae, and in osteoblast-enriched (ob) cultures isolated from fetal rat parietal bone. In cultured calvariae, 0.5-5 nM PTH-rp stimulated [3H]thymidine incorporation into DNA by 25-70% after 24 h of treatment and decreased relative [3H]proline incorporation into collagen by 50%; the inhibitory effect on collagen production was not altered by hydroxyurea, which decreased DNA synthesis by 85%. PTH-rp also increased [3H]hydroxyproline levels by 100% in culture medium from bones prelabeled with [3H]proline, indicating accelerated matrix turnover. In contrast to results in intact calvariae, PTH-rp had little effect on basal DNA and collagen synthesis in serum-deprived ob cultures. However, when ob cultures were treated with transforming growth factor type beta at concentrations similar to those found in calvarial culture medium, 0.02-2 nM PTH-rp enhanced DNA synthesis and decreased collagen production. Furthermore, equimolar PTH-rp and PTH concentrations similarly displaced 125I-PTH-rp binding and enhanced cAMP synthesis in ob cultures. These studies suggest that PTH-rp regulates osteoblastic cell activity primarily through PTH-related pathways and may act in part by modulating the effects of locally produced transforming growth factor-beta in bone.

Growth stimulation of 3T3 fibroblasts by Cystatin

Treatment of cultures of mouse 3T3 fibroblasts with Cystatin C, a thiol-proteinase inhibitor isolated from chicken egg white, resulted in an enhanced rate of cell proliferation. This stimulation was demonstrated using two independent assay systems: (a) assessment of total cell number and (b) measurement of [ 3H]thymidine incorporated into acid-precipitable DNA. In both assays, the dose-response curves of Cystatin stimulation showed a rising function that plateaued at a concentration of ~120 μg/ml. The addition of Cystatin to cultures of Kirsten murine sarcoma virus-transformed 3T3 cells also enhanced DNA synthesis in these target cells. Control experiments showed that the presence of Cystatin did not alter the level of binding of radioactively labeled epidermal growth factor and platelet derived growth factor to 3T3 cells. These results argue against the possibility that the observed growth stimulation by Cystatin was due to growth factor contamination of the Cystatin preparation.

Inhibition of epidermal growth factor-mediated DNA synthesis by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the spontaneously hypertensive rat

Aortic vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) grow nearly twice as fast in vitro as cells isolated from several normotensive control strains of rats. We have previously shown that DNA synthesis in SHR cells from both young and adult animals in response to epidermal growth factor is selectively enhanced compared with normotensive controls, suggesting that epidermal growth factor may be at least partly responsible for the enhanced growth rate. To determine whether the enhanced DNA synthesis in response to epidermal growth factor in SHR cells is mediated via an enhanced epidermal growth factor receptor tyrosine kinase, we measured thymidine incorporation in epidermal growth factor-stimulated vascular smooth muscle cells in the presence of the highly specific tyrosine kinase inhibitor genistein. The 50% inhibitory dose (IC50) of genistein was higher for the SHR vascular smooth muscle cells than for the normotensive Wistar rat (NBR; National Institutes of Health Black rat). This suggests that the increased DNA synthesis in response to epidermal growth factor in SHR cells is a result of higher receptor tyrosine kinase activity initiating further intracellular signals.

Differential response of normal human fibroblasts to bombesin versus thrombin.

Normal human diploid fibroblasts (WS-1 cells) were growth-arrested under serum-free conditions for 48 hr. The addition of fetal bovine serum (10% final concentration) to these cells stimulated [3H]-thymidine incorporation into DNA and phosphoinositide breakdown over nine-fold. Thrombin, at concentrations above 0.1 unit/ml (u/ml), was also effective at stimulating DNA synthesis and phosphoinositide breakdown as well as causing a rise in intracellular pH. In contrast, the peptide bombesin (concentrations ranging from 1 nM to 100 nM) stimulated phosphoinositide breakdown but did not enhance DNA synthesis or cause an increase in cytoplasmic pH. The time course of accumulation of inositol phosphates differed in response to these agents. The thrombin effect peaked rapidly and leveled off after 5 min while the bombesin effect showed a constant increase for 30 min. Serum showed an intermediate response. The different rates of inositol phosphate accumulation observed with the two growth factors is viewed as representing a difference in the mechanism of phosphoinositide turnover. The relationship between the difference in phosphoinositide turnover and the initiation of DNA synthesis is also discussed.

Lack of inhibition of DNA synthesis by prostaglandin I 2 in cultured intimal smooth muscle cells from rabbits

The negative control system for proliferation by prostaglandin I 2 (PGI 2) was studied in the smooth muscle cells (SMC) cultured from the thickened intima (intimal SMC) of rabbit aortas. Indomethacin was found to enhance DNA synthesis of medial SMC but not that of intimal SMC. Exogenously added PGI 2 or its stable analogue, CS-570, was observed to inhibit DNA synthesis of medial SMC enhanced by indomethacin but not that of intimal SMC. These results indicate that medial SMC are negatively controlled by endogenous PGI 2 and that intimal SMC have no such negative control system for cell proliferation. This lack of negative control may be one of the mechanisms underlying the rapid growth behavior of intimal SMC as compared to medial SMC.

In vitro lymphocyte proliferation by carbamazepine, carbamazepine-10, 11-epoxide, and oxcarbazepine in the diagnosis of drug-induced hypersensitivity

In vitro lymphocyte proliferation induced by carbamazepine (CBZ) was evaluated in nine patients with hypersensitivity to this drug. Lymphocytes from all hypersensitive patients responded by significantly enhanced DNA synthesis to CBZ when patients were compared with 33 tested control subjects. However, lymphocytes from five of six hypersensitive patients were not stimulated by carbamazepine-10, 11-epoxide, and oxcarbazepine (OXC), and this was confirmed clinically in two CBZ hypersensitive patients with OXC therapy. The results indicate that OXC may be suitable as an alternative therapy in some patients with CBZ hypersensitivity.

Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I 2 in cultured rabbit aortic smooth muscle cells

The role of prostaglandin 12 (PGI 2) in the control of DNA synthesis during the cell cycle was investigated in cultured rabbit aortic smooth muscle cells (SMC). SMC at confluency in the G 0 state reached the S phase about 16 h after stimulation with serum, as judged by measurement of [ 3H]thymidine incorporation into DNA (DNA synthesis). Cyclooxygenase inhibitors such as indomethacin and aspirin enhanced DNA synthesis, suggesting that endogenously synthesized prostaglandins inhibit DNA synthesis. Added PGE 1 or PGE 2 had little effect on DNA synthesis. PGI 2 inhibited DNA synthesis only when added from 10 to 16 h after stimulation of SMC in the G 0 state with serum. Addition of CS-570, a stable PGI 2 analogue, inhibited DNA synthesis at any time after serum stimulation. The endogenous syntheses of (PGI 2) and DNA were negatively correlated. These results suggest that PGI 2 inhibits DNA synthesis by acting on the progression stage of the G 1 state.

Comparative effects of tumor necrosis factor-alpha and IL-1 beta on mitogen-induced T cell activation.

The effect of rTNF-alpha on human T cell function was examined and compared with that of rIL-1 beta by assessing the ability of each cytokine to support mitogen-induced proliferation, IL-2 production, and IL-2R expression. TNF-alpha and IL-1 beta each enhanced DNA synthesis induced by PHA or immobilized mAb to the CD3 molecular complex. In addition, each cytokine increased the number of cells entering the G1 phase of the cell cycle and augmented IL-2R expression. The combination of optimal concentrations of these factors supported these responses to a greater extent than either cytokine alone, suggesting that T cell responsiveness is independently regulated by the action of at least two separate monocyte derived cytokines. Whereas TNF-alpha had little effect, IL-1 beta augmented IL-2 mRNA expression and IL-2 production by mitogen-stimulated cells. Furthermore, IL-1 beta enhanced proliferation with increasing length of culture. Whereas TNF-alpha also enhanced proliferation late in culture, it was less effective in this regard than IL-1 beta. Thus, IL-1 beta and TNF-alpha augment mitogen-induced T cell proliferation by increasing the number of cells initially activated and by promoting subsequent cell cycle progression. They differ, however, in their capacity to promote IL-2 mRNA and IL-2 production and therefore ongoing T cell proliferation.

The effects of hydrocortisone on c- fos, c- myc and c- ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c- fos expression during the G 1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c- fos expression. Quiescent cells showed no change in c- fos expression during the G 1 phase of the cell cycle. Three peaks of c- fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c- myc or c-Ha- ras expression in the presence or absence of serum is sybchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c- fos and c- myc, and the cytoplasmic oncogene c- ras are independent and hydrocortisone may enhance DNA synthesis by increasing c- fos expression.

Biostimulation of HeLa cells by low-intensity visible light. V. Stimulation of cell proliferationin vitro by He−Ne Laser irradiation

Autoradiographic experiments, performed with monolayer HeLa cells, show that the irradiation with He-Ne laser (D=100 J/m2) causes an increase in the number of S-phase cells connected with enhanced G1-S transition of a part of the population, as well as an increase in the grain count on the labelled nuclei connected with an enhancement of DNA synthesis in Sphase cells. The irradiation influences the proliferation rate of various subpopulations not in equal degree, as shown analysing the clone size distribution after the irradiation (D=10, 102, 103J/m2). The stimulative effect of irradiation is most noticeable on the proliferation activity of the slowly growing subpopulations.

The differential effect with time of neuropeptides on the proliferative responses of murine Peyer's patch and splenic lymphocytes

The neuropeptides substance P (SP), somatostatin (SOM), and vasoactive intestinal peptide (VIP) have been shown to modulate lymphocyte DNA, RNA, and immunoglobulin synthesis. We have previously shown that SP enhances while SOM and VIP inhibit proliferation of murine splenic and Peyer's patch lymphocytes when cells were cultured with concanavalin A and neuropeptides for 72 h. Here we show that the effect of neuropeptides, in particular SP, is dependent on the amount of time that lymphocytes are incubated with NP. We found that SOM and VIP always inhibited cell proliferation for incubation times of 2 to 72 h. In contrast when cells were exposed to SP for 24 h or less, there was inhibition of [ 3H]thymidine uptake by both Peyer's patches and splenic lymphocytes. Significant enhancement in DNA synthesis by lymphocytes from both organs was only seen when cells were incubated with SP for the whole 72 h. We believe that our data may explain some of the conflicting reports regarding the effects of neuropeptides on cell proliferation.

Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells.

Human peripheral blood, spleen, and lymph node B cells were stimulated with Cowan I Staphylococcus aureus (SA) or F(ab')2 fragments of anti-mu antibody (anti-mu) and various lymphokines and were analyzed for proliferation and generation of Ig-secreting cells (ISC). SA alone but not anti-mu stimulated minimal proliferation of each population. Recombinant IL 2 (r-IL 2) effectively promoted proliferation of SA-stimulated blood and spleen B cells, but supported less vigorous responses of lymph node B cells. By contrast, r-IL 2 enhanced DNA synthesis of all anti-mu-stimulated B cells early in culture, but did not promote sustained proliferation of anti-mu-stimulated lymph node B cells and only promoted ongoing DNA synthesis of some anti-mu-activated blood (eight out of 17) and spleen (five out of 14) B cell preparations. Recombinant interferon-gamma (r-IFN-gamma) and a commercial preparation of B cell growth factor (BCGF) also augmented DNA synthesis of all three B cell populations stimulated with SA or anti-mu early in culture, but neither alone was able to sustain maximal proliferation. Markedly enhanced sustained proliferation of all three anti-mu- and SA-stimulated B cell populations was noted when cultures were supported by the combination of r-IL 2 and BCGF, or to a lesser extent by r-IL 2 and r-IFN-gamma. The generation of ISC from SA-stimulated blood or spleen but not lymph node B cells was effectively supported by r-IL 2 alone. Differentiation of lymph node B cells required the combination of r-IL 2 and BCGF. These studies emphasize the importance of both the activation stimulus and the origin of the B cells in determining the lymphokine requirements of human B cell responsiveness.

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells.

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.

Effect of histamine H 2-receptor antagonists on DNA synthesis in adult rat hepatocytes in primary culture. Cimetidine enhanced hepatocytes proliferation stimulated with insulin and epidermal growth factor

The effects of three of the most widely used histamine H 2-receptor antagonists, cimetidine, ranitidine and famotidine, on liver cell growth were studied in vitro using adult rat hepatocytes in primary culture, because these antagonists are commonly given to patients with hepatic cirrhosis or fulminant hepatic failure for protection against peptic ulcers and gastrointestinal hemorrhage. At their clinically effective concentrations in the blood (0.5–5 μg/ml cimetidine, 0.25–2.5 μg/ml ranitidine and 0.05–0.5 μg/ml famotidine), these three antagonists did not have any effect on replicative DNA synthesis either in the presence or absence of insulin plus epidermal growth factor (EGF). However, unexpectedly DNA synthesis stimulated by insulin and EGF was found to be enhanced by 0.05–0.5 mg/ml cimetidine, although it was unaffected or inhibited by ranitidine and famotidine at the concentrations tested. Cimetidine caused maximal enhancement of 1.5–2 times the control level of DNA synthesis at a concentration of 0.25 mg/ml. Cimedidine also had an enhancing effect at submaximal concentrations of insulin and EGF, but neither cimetidine nor the other antagonists had any stimulatory effect on DNA synthesis in the absence of insulin plus EGF. This enhancement of DNA synthesis by cimetidine resulted in significant increase in the total DNA content of the hepatocytes in culture. Under the conditions used, cimetidine had the lowest toxicity of these three antagonists and ranitidine the highest, as judged from data on DNA synthesis and the total protein content of cultured hepatocytes, leakage of aminotransferases from the cells and morphological observations.