Stimulation Of Deoxyribonucleic Acid Synthesis Research Articles

Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells

A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5′-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L–10 μmol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine ≫ isoproterenol, indicating that α adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by α1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by α1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 μmol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via α1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.

Prostaglandins mediate the stimulation of deoxyribonucleic acid synthesis by transforming growth factor alpha in hen granulosa cells during ovarian follicular development.

The present study was an examination of the possible involvement of specific membrane lipid metabolites of prostaglandins (PGs), leukotrienes (LTs), lysophosphatidic acid (LPA), and lysophosphatidyl choline (LPC) in transforming growth factor alpha-(TGF alpha) induced DNA synthesis by granulosa cells during follicular development. Granulosa cells from the first (F1) and the fifth and sixth (F5-6) largest preovulatory follicles were cultured for 18 h in the presence of TGF alpha and/or an inhibitor of either phospholipase A2 (PLA2), cyclooxygenase, or lipoxygenase, LTs, LPA, LPC, and/or PGs. TGF alpha-induced, but not basal, DNA synthesis in F1 and F5-6 granulosa cells was suppressed by inhibitors of PLA2 and cyclooxygenase but not of lipoxygenase. The inhibition was concentration-dependent and could be attenuated by exogenous PGE2. Likewise, PGF and PGE production was suppressed by these inhibitors. Moreover, PGE2 and, to a lesser extent, PGF2 alpha increased basal [3H]thymidine incorporation and enhanced DNA synthesis induced by a submaximal stimulatory concentration of TGF alpha. The mitogenic effect of PGs was more evident in granulosa cells from F5-6 follicles than in those from F1 follicles. In contrast, LTs (LTB4, LTC4, LTD4), (+)5(s)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE), and lysophospholipids (LPA and LPC) had no effect on granulosa cell DNA synthesis, irrespective of the stage of ovarian follicular development and the presence of TGF alpha and PGE2. These studies demonstrate that 1) arachidonic metabolites of cyclooxygenase, but not lipoxygenase pathway and arachidonate lysophospholipid by-products (LPA and LPC), are involved in the regulation of granulosa cell DNA synthesis by TGF alpha; 2) compared to PGs of the F series, PGE2 and PGE1 are more effective in either mimicking the action of TGF alpha or potentiating the mitogenic response of the cells to the growth factor; and 3) the DNA synthetic response of the hen granulosa cell to PGs is greater in granulosa cells from F5-6 than in those from F1 follicles. These findings are consistent with our concept that an increase in PG production is a necessary element in the TGF alpha-regulated cascade of biochemical events leading to chicken granulosa cell mitogenesis during hen ovarian follicular development.

Expression of a functional endothelin (ETA) receptor in human meningiomas

Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 +/- 0.3 nM, maximum binding capacity: 319 +/- 66 fmol/mg (mean +/- standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 +/- 0.7 x 10(-9) M, whereas ET-3 showed a much lower affinity (IC50: 8.4 +/- 2.5 x 10(-6) M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10(-7) M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphate-ribose to the alpha subunit of guanine nucleotide binding (G) proteins such as Gi or G(o), induced a concentration-dependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.

Prostaglandin E 2 antagonizes gingival fibroblast proliferation stimulated by interleukin-1β

Treatment of human gingival fibroblasts with prostaglandin E 2 (PGE 2) resulted in significant concentration-dependent inhibition in deoxyribonucleic acid (DNA) synthesis (8.40–37.89%), while indomethacin (INDO) (PG inhibitor), interleukin-1β (IL-1β) or IL-1β + INDO caused a significant and dose-dependent increase in DNA synthesis. Addition of PGE 2 to culture media containing IL-1β and INDO caused a significant concentration-dependent reduction in IL-1β- and INDO-induced stimulation of DNA synthesis. The findings suggest that IL-1β and PGE 2, which are also produced by fibroblasts, could play an important role in regulation of gingival tissue development and wound healing, and their modulation may have therapeutic potential.

Characterization of a mouse hepatocyte growth-stimulating factor in serum of mice treated with carbon tetrachloride.

The physicochemical and biological properties of a mouse hepatocyte growth-stimulating factor (mHGSF), whose amount in mouse serum increased markedly 24 h after carbon tetrachloride administration (E. Gohda et al., Life Sci. 46, 1801 (1990)), were examined. This factor was a heat-labile protein with a molecular weight of 75000. Its activity was sensitive to disulfide reduction. Maximal stimulation of deoxyribonucleic acid synthesis in cultured rat hepatocytes by this factor was greater than that by acidic fibroblast growth factor (acidic FGF) or mouse epidermal growth factor (EGF) and was comparable to maximal stimulation by human hepatocyte growth factor (hHGF), a heterodimer with a molecular weight of about 85000. The effect of mHGSF was additive to the maximal effects of acidic FGF and EGF and was synergistic with the maximal effect of insulin, but was neither additive nor synergistic with the maximal effect of hHGF. The mHGSF activity was not inhibited by a neutralizing anti-hHGF antiserum, which recognizes nonreduced hHGF but not reduced heavy and light chains of hHGF. mHGSF did not show any cross-reactivity to anti-hHGF monoclonal and/or polyclonal antibodies as measured by an enzyme-linked immunosorbent assay for hHGF. These results suggest that mHGSF is a hHGF-like factor with some structural difference from hHGF.

Purification and characterization of a mouse hepatocyte growth-stimulating factor from the liver of carbon tetrachloride-treated mice.

Mouse hepatocyte growth-stimulating factor (mHGSF), which increased markedly in the liver of carbon tetrachloride-treated mice, was purified 275,000-fold with 21% yield from extracts of the injured liver. The purification involves ammonium sulfate precipitation and chromatography on Affi-Gel Blue, heparin-Sepharose, and S-Sepharose. The purified factor migrated as a major band of 76,000 daltons under nonreducing conditions and two bands of 62,000 and 31,000 daltons under reduced conditions. A dose-response of this growth factor for stimulation of deoxyribonucleic acid synthesis in cultured rat hepatocytes and its maximal effects were similar to those of human hepatocyte growth factor (hHGF), which we previously purified from the plasma of patients with fulminant hepatic failure (E. Gohda et al., J. Clin. Invest., 81, 414-419 (1988)). The effect of mHGSF was additive to the maximal effect of epidermal growth factor and was synergistic with that of insulin or acidic fibroblast growth factor, but was neither additive nor synergistic with the maximal effect of hHGF. mHGSF, like hHGF, was sensitive to heat and trypsin treatments and to reduction by dithiothreitol. This factor did not react with an anti-hHGF antiserum. These results indicate that mHGSF is a hHGF-like factor, but it is immunologically different from hHGF.

The effects of an atherogenic diet on macrophage/biomaterial interactions

We previously reported that biomaterials differentially induce macrophages to secrete growth factors that mediate reendothelialization. The present study evaluates the effect of an atherogenic diet on macrophage/biomaterial interactions. Female New Zealand white rabbits were fed an atherogenic diet. Peritoneal macrophages were harvested from these as well as rabbits fed a normal diet and cultured in Minimum Essential Medium with platelet-poor serum. Dacron or polyglactin 910 were added to two of three conditions of both cell groups in passage 2. Conditioned media were collected weekly through week 15. Mitogenicity assays were performed with quiescent mouse embryonal (BALB/c3T3) fibroblasts, atherosclerotic rabbit aortic smooth muscle cells, and murine capillary lung (LE-II) endothelial cells. Mitogenic activity was assayed by scintillation counting of tritiated thymidine incorporation into deoxyribonucleic acid (DNA). Results showed increased mitogenic activity released by macrophages from atherosclerotic rabbits, in the absence of prosthetic material, when assayed against every cell line. In normal diet macrophages, polyglactin 910 stimulated mitogen release for every cell line, and Dacron yielded minimal mitogen release. In lipid diet macrophages polyglactin 910 slightly increased mitogen release for all three cell lines, whereas Dacron resulted in stimulation of DNA synthesis in smooth muscle cells and BALB/c3T3 cells but less DNA synthesis in LE-II cells than in control, no graft material, media. Western blotting demonstrated immunoreactivity to basic fibroblast growth factor in media from normal diet macrophages but only in the presence of polyglactin 910 or Dacron. Radioimmunoassay for platelet-derived growth factor B chain was negative in all groups, and polymerase chain reaction techniques to amplify transforming growth factor-beta messenger ribonucleic was negative. These data demonstrate the effect of in vivo dietary manipulation on macrophage activation as well as the effect of an atherogenic diet in modulating macrophage/biomaterial interactions. Additionally, different biomaterials differentially induce macrophages to release factors that stimulate and inhibit growth.

The effects of an atherogenic diet on macrophage/biomaterial interactions

We previously reported that biomaterials differentially induced macrophages to secrete growth factors that mediate reendothelialization. The present study evaluates the effect of an atherogenic diet on macrophage/biomaterial interactions. Female New Zealand white rabbits were fed an atherogenic diet. Peritoneal macrophages were harvested from these as well as rabbits fed a normal diet and cultured in Minimum Essential Medium with platelet-poor serum. Dacron or polyglactin 910 were added to two of three conditions of both cell groups in passage 2. Conditioned media were collected weekly through week 15. Mitogenicity assays were performed with quiescent mouse embryonal (BALB/c3T3) fibroblasts, atherosclerotic rabbit aortic smooth muscle cells, and murine capillary lung (LE-II) endothelial cells. Mitogenic activity was assayed by scintillation counting of tritiated thymidine incorporation into deoxyribonucleic acid (DNA). Results showed increased mitogenic activity released by macrophages from atherosclerotic rabbits, in the absence of prosthetic material, when assayed against every cell line. In normal diet macrophages, polyglactin 910 stimulated mitogen release for every cell line, and Dacron yielded minimal mitogen release. In lipid diet macrophages polyglactin 910 slightly increased mitogen release for all three cell lines, whereas Dacron resulted in stimulation of DNA synthesis in smooth muscle cells and BALB/c3T3 cells but less DNA synthesis in LE-II cells than in control, no graft material, media. Western blotting demonstrated immunoreactivity to basic fibroblast growth factor in media from normal diet macrophages but only in the presence of polyglactin 910 or Dacron. Radioimmunoassay for platelet-derived growth factor B chain was negative in all groups, and polymerase chain reaction techniques to amplify transforming growth factor-beta messenger ribonucleic was negative. These data demonstrate the effect of in vivo dietary manipulation on macrophage activation as well as the effect of an atherogenic diet in modulating macrophage/biomaterial interactions. Additionally, different biomaterials differentially induce macrophages to release factors that stimulate and inhibit growth.

Enhancing effect of S-(1,2-dicarboxyethyl)glutathione on epidermal growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes

A tripeptide S-(1,2-dicarboxyethyl)glutathione (DCE-GS) has been reported to be present in the lens, liver, and heart. Effects of DCE-GS and its derivatives and analogues on hepatocyte deoxyribonucleic acid (DNA) synthesis were examined using primary cultures of adult-rat hepatocytes. DCE-GS alone had no effect on DNA synthesis of hepatocytes. However, when DCE-GS was added with epidermal growth factor (EGF), the tripeptide effectively enhanced EGF-stimulated DNA synthesis of hepatocytes under the culture conditions of low cell density, but not high cell density. On the other hand, some esters and amides of DCE-GS and DCE-GS analogues showed a suppressive effect on DNA synthesis of hepatocytes in the absence of EGF. The derivatives and analogues together with EGF had no effect or rather a suppressive effect on stimulation of hepatocyte DNA synthesis by EGF. Therefore, the two carboxy groups in the substituent probably play an important role in the stimulative activity of DCE-GS. In addition, it seems likely that one of in vivo physiological roles of DCE-GS is related to liver regeneration.

Lignified materials as potential medicinal resources. III. Diversity of biological activity and possible molecular species involved.

Diverse biological activities of hot-water and alkali extracts of lignified materials were reviewed and the molecular species involved are discussed. Materials tested included pine cone of Pinus parviflora SIEB. et Zucc., wood chips of slash pine, Douglas fir, and tallow wood, and two basidiocarps, in addition to their partially degraded preparations and commercial lignins. As a tentative conclusion, the lignin structure of these extracts might be responsible for the potent stimulation of granulocytic cell iodination, inhibition of viral infection and/or proliferation in vitro, and inactivation of viral ribonucleic acid (RNA)-dependent RNA polymerase and (adenosine diphosphate-ribose)n glycohydrolase. Other activities displayed by some of these extracts, such as antibacterial and antitumor activities, induction of hemolytic plaque-forming-cells in mice, and stimulation of deoxyribonucleic acid synthesis of isolated splenocytes, remain to be investigated.

Supranormal stimulation of deoxyribonucleic acid synthesis in FRTL5 cells by serum from patients with untreated acromegaly.

Insulin-like growth factor I (IGF-I) is one of a number of mitogenic factors in the serum of animals and humans. We demonstrated previously that IGF-I is a potent mitogen for FRTL5 cells, a line of rat thyroid follicular cells. In this study, we assessed the relevance of this finding with respect to the levels of IGF-I found in human serum by comparing the effects of normal serum and serum from patients with untreated acromegaly or hypopituitarism on DNA synthesis in quiescent FRTL5 cells. As expected, when added to cells maintained in Coon's modified Ham's F-12 medium containing 0.1% BSA, but devoid of insulin, transferrin, TSH, or calf serum, normal serum produced a dose-dependent stimulation of [3H]thymidine incorporation into DNA. A similar, but more marked, effect was produced by the addition of serum from patients with untreated acromegaly. In multiple experiments, a standard concentration (0.5%) of acromegaly serum was more stimulatory to DNA synthesis than was normal serum. In a single experiment designed to eliminate interassay variation and define the relationship between the ability of serum to stimulate DNA synthesis and its IGF-I concentration, studies were performed with 0.5% concentrations of serum from 9 normal subjects, 15 patients with untreated acromegaly, and 3 patients with panhypopituitarism. On the average, [3H]thymidine incorporation in the presence of serum from patients with acromegaly was approximately 3 times, and that in the presence of serum from patients with hypopituitarism only one fourth, that in the presence of serum from normal subjects; there was no overlap of individual values in the three groups. For the entire study group, we found a highly significant correlation (r = 0.86) between the serum IGF-I concentration and the ability of that serum to stimulate thymidine incorporation into the DNA of FRTL5 cells. The stimulatory effects of serum from both normal and acromegalic subjects were decreased or abolished by the addition of a monoclonal antibody against IGF-I. In hypophysectomized rats, GH increases the thyroid to body weight ratio and enhances the effect of TSH to promote thyroid growth. Further, an inordinate frequency of nontoxic goiter in patients with acromegaly has been reported. Taken together with these observations, our findings suggest that the effect of IGF-I to promote thyroid cell growth in vitro has a counterpart in the living animal or patient.

Progesterone and dexamethasone inhibition of uterine epithelial proliferation in two models of estrogen-independent growth

It is believed that progestins and glucocorticoids block estrogen-induced growth of the uterus by perturbing the mechanism of estrogen stimulation. We have examined the effect of progesterone or dexamethasone on two rodent models of estrogen-independent proliferation of uterine epithelium. Tissues of the neonatal mouse uterus exhibit high rates of deoxyribonucleic acid synthesis in the absence of endogenous steroids. Progesterone and dexamethasone inhibit deoxyribonucleic acid synthesis in the uterine epithelium of 4-day-old mice. When grafted beneath the kidney capsule of an ovariectomized adult, the tissues of the neonatal uterus continue to grow without the addition of estrogen. Both progesterone and dexamethasone blocked uterine epithelial mitotic activity in these allografts. A true growth response can be stimulated in the uterus of an ovariectomized rat by intraluminal instillation of cholera toxin. In the present study, autoradiographic analysis showed that cholera toxin stimulation of deoxyribonucleic acid synthesis was confined to the epithelial compartment in ovariectomized, vehicle-treated rats. When ovariectomized rats were pretreated with progesterone, cholera toxin induced deoxyribonucleic acid synthesis in the stroma but not in the epithelium. Thus progesterone and dexamethasone inhibit uterine epithelial proliferation that is not driven by estrogen stimulation. Therefore in these estrogen-independent model systems, the inhibitory effects of these steroids cannot be due to antagonism of estrogen receptor—mediated events. These observations suggest that inhibition in estrogen-induced uterine epithelial proliferation may also work through a mechanism unrelated to antagonism of estrogen receptor action.

Estrogen stimulation of deoxyribonucleic acid synthesis in uterine epithelial cells which lack estrogen receptors.

Autoradiographic studies from this laboratory have previously indicated that the uterine epithelium of the neonatal mouse is devoid of estrogen receptors. The neonatal murine uterus is composed of an undifferentiated mesenchyme and a simple columnar epithelium lining the lumen. In the present study, a method for measuring whole cell uptake of 16 alpha-[125I]iodoestradiol ([125I]iodo-E2) was developed and applied to enzymatically separated, isolated epithelial and mesenchymal cells of neonatal (4-5 days postnatal) uteri. Epithelial cells and fibromuscular cells (stromal and myometrial cells) from uteri of 21-day-old animals were used to validate the assay method. A component of the uptake of [125I]iodo-E2 by cells from 21-day-old uteri was shown to be specific, saturable, and of high affinity. Kd values for specific uptake by uterine mesenchymal and epithelial cells were 1.2-1.3 nM. Maximal specific uptake was 9.3 and 4.2 fmol/micrograms DNA for epithelial and mesenchymal cells, respectively. The uterine epithelial cells of 4- and 5-day-old mice showed no measurable specific uptake of [125I]iodo-E2, while the mesenchymal cells from these animals had a maximal specific uptake of 7.9 fmol/micrograms DNA, with a Kd of 1.3 nM. DNA synthesis increased within the uterine epithelium of neonatal mice after estrogen treatment. The thymidine labeling index was doubled 10 h after a single dose of diethylstilbestrol (DES) and returned to pretreatment values by 18 h. The epithelial mitotic index was also 2-fold higher than control values 16-18 h after DES treatment. The increase in the thymidine labeling index was specific to estrogen treatment. DES did not induce the production of estrogen receptors in neonatal uterine epithelium. Epithelial cells of 5-day-old mice that were treated with DES showed no specific [125I]iodo-E2 uptake, while whole cell uptake by mesenchymal cells from these animals exhibited a specific, high affinity component, with a maximal binding of 8.4 fmol/micrograms DNA. Autoradiographic analysis of [3H]estradiol uptake by uterine tissues from 5-day-old mice 12 h after DES treatment did not show nuclear concentration of the steroid in the epithelial cells. These results indicate that the uterine epithelium of the neonatal mouse is indeed devoid of estrogen receptors, and yet the rate of DNA synthesis within this tissue is responsive to estrogen stimulation. The epithelial cells remain devoid of estrogen receptors after DES stimulation, indicating that intraepithelial estrogen receptors are not required for induction of DNA synthesis in these cells in situ. Possible mechanisms by which this phenomenon may occur are discussed.

16,16-Dimethyl prostaglandin E2 suppresses the increases in the proliferative activity of rat colonic epithelium induced by indomethacin and aspirin

Treatment of rats with indomethacin rapidly increased ornithine decarboxylase (4 h) of colonic mucosa and [3H]thymidine incorporation into coJonic mucosal deoxyribonucleic acid (DNA) (1 or 5 days) when this parameter was examined in vivo and ex vivo. The changes in colonic mucosal ornithine decarboxylase and DNA synthesis induced by indomethacin were correlated temporally with suppression of colonic prostaglandin synthesis, as assessed from ex vivo colonic production of prostaglandin E, the dominant prostaglandin product of colon. Autoradiographic studies indicated that the enhancement of proliferative activity of colonic epithelium after treatment with indomethacin for 1 day was confined to the lower third of the colonic crypt (normal proliferative zone). After 5 days of indomethacin treatment, however, there was an extension of the proliferative zone to the upper third of the colonic crypts. Concurrent treatment of rats with the stable prostaglandin E2 analogue, 16,16-dimethyl prostaglandin E2, suppressed indomethacin-induced increases in colonic mucosal ornithine decarboxylase and DNA synthesis. Concurrent administration of 16,16-dimethyl prostaglandin E2 also prevented the extension of the proliferative zone of colonic epithelium induced by 5 days of indomethacin administration. 16,16-Dimethyl prostaglandin E2 alone for 1–5 days had no detectable effects on colonic mucosal ornithine decarboxylase and DNA synthesis compared with corresponding control values. Increases in colonic mucosal DNA synthesis were also induced by treatment of rats for 5 days with aspirin (ASA). The stimulation of colonic mucosal DNA synthesis induced by ASA was significantly suppressed by concurrent administration of 16,16-dimethyl prostaglandin E2 and was also correlated with the inhibition of colonic prostaglandin synthesis by ASA. The colons of rats treated with indomethacin for 1 day or ASA for 5 days appeared normal by light microscopy. However, treatment of rats for 5 days with indomethacin resulted in mild to moderate inflammation of the lamina propria and some goblet cell depletion at the mucosal surface, but no loss of surface epithelium. The ultrastructure of the surface epithelium of the colons of rats treated with indomethacin or ASA was normal as assessed by electron microscopy. The results thus demonstrate that inhibition of local colonic prostaglandin synthesis is associated with increases in the proliferative activity of colonic epithelium, and that these increases are suppressed by administration of 16,16-dimethyl prostaglandin E2. Under some conditions, the increases in proliferative activity that were observed with indomethacin and ASA are dissociated from drug-induced inflammation and cell surface injury.

Aging alters somatomedin-C-dexamethasone synergism in the stimulation of deoxyribonucleic acid synthesis and replication of cultured human fibroblasts.

The effects of dexamethasone on somatomedin-C (SM-C) stimulation of [3H]thymidine incorporation and cell replication were studied in early passage fibroblasts from normal donors, aged 7-24 yr (young) and 85-96 yr (old), and one patient with progeria. Preincubation of cells from young donors with dexamethasone dramatically enhanced SM-C stimulation of [3H]thymidine incorporation [e.g. 19- vs. 3-fold in serum-free medium; 66- vs. 14-fold in 0.25% human hypopituitary serum (HHS)], with no alteration in the timing of peak thymidine incorporation. In contrast, preincubation of cells from old and progeric donors with dexamethasone resulted in a 6- to 12-hr lengthening of the prereplicative period and, generally, little or no synergism with SM-C. Cells from old and progeric donors had a normal replicative response to SM-C with or without 0.25% HHS. In cells from young donors, dexamethasone enhanced the SM-C-stimulated increase in cell number 32-49% in serum-free medium and 70-189% in 0.25% HHS. In comparison, dexamethasone had no potentiating effect on SM-C stimulation of multiplication of cells from old and progeric donors. These data indicate that dexamethasone and SM-C are synergistic in stimulating DNA synthesis and replication of fibroblasts from young donors, but that this synergism is impaired in cells from aged and progeric donors.

Evidence for a discontinuous requirement for estrogen in stimulation of deoxyribonucleic acid synthesis in the immature rat uterus.

Estriol is rapidly lost from uterine target cell nuclei. Therefore, it has been used to study the relative time of exposure to estrogen that is necessary to elicit the late growth responses of increased DNA polymerase activity or increased rate of DNA synthesis observed after 17beta-estradiol treatment. Estriol administered during a second critical phase from 9-15 h after the initial injection elicits maximal DNA synthesis. Our results suggest that there is a discontinuous requirement for estrogen in the sequence of events which result in the uterine growth response to estrogenic hormones. If estriol is present during critical phases of this sequence of events, it is equipotent to estradiol in eliciting the full uterotropic response.

The correlation of the ongoing proliferative response to tumour in lymphoid organs with the anti-tumour activity of the lymphoid cells

Anti-tumour activity of washed lymph node (LN) and spleen cells of mice bearing a transplanted methylcholanthrene-induced tumour (MC 1) for 10–14 or 25–28 days was compared and correlated with the stimulation of deoxyribonucleic acid (DNA) synthesis in the lymphoid organs. While the anti-tumour effect per lymph node cell (LNC) changed little if at all during this period, the stimulation of DNA synthesis in the total cell population of the nodes diminished significantly by the 25–28 day stage of tumour-bearing in contrast to the markedly high level seen at 10–14 days. In the spleen, however, stimulation of DNA synthesis in the total cell population increased throughout the period of study while the anti-tumour activity per cell significantly decreased. Depressed effector activity of spleen cells was shown to be associated with a decrease in anti-tumour activity of the T cell population. These observations provide evidence for a suppression of the immune response to tumour antigens operating at the level of sensitization of the immunocompetent cells and further suggest that proliferation in the tumour-bearing host of a cell clone(s) with suppressor function may have a direct or indirect role in controlling the specific response to tumours.

Mitogenic response of mouse spleen cells and gelation of limulus lysate by lipopolysaccharide of Yersinia pestis and evidence for neutralization of lipopolysaccharide by polymyxin B

Lipopolysaccharide (LPS) extracted with phenol and water from Yersinia pestis was compared with LPS of Escherichia coli for stimulation of deoxyribonucleic acid synthesis in mouse spleen cells (lymphocyte mitogenesis), gelation of limulus lysate, pyrogenicity in the rabbit, and susceptibility to inhibition of these activities by polymyxin B sulfate (PBS). LPS of Y. pestis stimulated deoxyribonucleic acid synthesis in mouse spleen cell cultures over the same quantitative range as LPS of E. coli. In the limulus tests and rabbit pyrogenicity studies, the LPS of Y. pestis was active but about 10 times less potent than E. coli LPS on a weight basis. PBS in concentrations from 1 to 10 microgram/ml diminished the rate of deoxyribonucleic acid synthesis in spleen cell cultures stimulated by LPS of both Y. pestis and E. coli. Addition of PBS to LPS of both Y. pestis and E. coli in a ratio of 100 parts of PBS to 1 part of LPS by weight increased by 10-fold the concentration of LPS required to produce gelation of limulus lysate and inhibited significantly pyrogenic responses in rabbits. These results demonstrating similarities of LPS of Y. pestis and E. coli may suggest that the pathogenesis of plague is similar to that of other gram-negative bacterial infections.

Neurotrophic stimulation of DNA synthesis in the regenerating forelimb of the newt, Triturus.

The effect of infusions of soluble newt brain extracts on deoxyribonucleic acid (DNA) synthesis in the denervated, regenerating newt blastema has been studied. When moderate early to early regenerates are denervated for 48 hours, DNA synthesis normally drops to about 40% of that found in contralateral, innervated blastemas. Infusion of brain extract halfway through the denervation period (at 24 hours) approximately doubles the rate of DNA synthesis in denervated regenerates.

Studies on the stimulation and suppression of deoxyribonucleic acid (DNA) synthesis in lymph node cells of mice bearing progressively growing tumours.

Host responsiveness to a progressively growing methylcholanthrene (MC) induced tumour (MC6/2) was studied at varying intervals following subcutaneous (s.c.) tumour implantation by monitoring the in vitro incorporation of tritiated thymidine (3H-TdR) into lymph node cells (LNC) undergoing stimulation in vivo and concurrently determining the total numbers of the lymphoid cells present in these organs at each of the time intervals. It was found that an initial period of rapidly increasing stimulation of DNA synthesis in lymph nodes was soon followed by the onset of a stage of decrease of this activity. Within limits, the larger the tumour inoculum the stronger the initial response. The suppression of stimulation of DNA synthesis that ensued appeared to be directly related to the tumour mass and to the dose of tumour cells implanted. The total numbers of the cells accumulating in nodes also increased initially but remained elevated during the subsequent period of tumour growth. Continued presence of the tumour was essential for the increased DNA synthesis in lymph nodes since tumour removal leads to a rapid decrease to levels found in tumour-free animals. These findings demonstrate that the failure to eradicate an antigenic tumour by its host may not be solely due to "desensitizing" and "blocking" factors but that other important mechanisms are also involved. We suggest that the inability to reject the tumour in this situation is dependent in considerable measure on the development of a state of hyporeactivity in the host due to the partial inhibition of the DNA synthetic response, possibly in T cells of the tumour host, due to "suppressor factor(s)" interacting with the immunocompetent cells.