Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Role of HIF-1alpha and VEGF in human mesenchymal stem cell proliferation by 17beta-estradiol: involvement of PKC, PI3K/Akt, and MAPKs

17beta-Estradiol (E(2)) is a steroid hormone well known for its roles in the regulation of various cell functions. However, the precise role that E(2) plays in the proliferation of human mesenchymal stem cells (hMSCs) has not been completely elucidated. In the present study, we examined the effects of E(2) on cell proliferation and the related signaling pathways using hMSCs. We showed that E(2), at > or =10(-9) M, significantly increased [3H]thymidine incorporation after 24 h of incubation, and E(2) also increased [3H]thymidine incorporation at >6 h. Also, E(2) significantly increased the percentage of the cell population in the S phase based on FACS analysis. Moreover, E(2) increased estrogen receptor (ER), PKC, phosphatidylinositol 3-kinase (PI3K)/Akt, and MAPK phosphorylation. Subsequently, these signaling molecules were involved in an E(2)-induced increase of [3H]thymidine incorporation. E(2) also increased hypoxia-inducible factor (HIF)-1alpha and VEGF protein levels. These levels of protein expression were inhibited by ICI-182,780 (10(-6) M, an ER antagonist), staurosporine and bisindolylmaleimide I (10(-6) M, a PKC inhibitor), LY-294002 (10(-6) M, a PI3K inhibitor), Akt inhibitor (10(-5) M), SP-600125 (10(-6) M, a SAPK/JNK inhibitor), and PD-98059 (10(-5) M, a p44/42 MAPKs inhibitor). In addition, HIF-1alpha small interfering (si)RNA and ICI-182,780 inhibited E(2)-induced VEGF expression and cell proliferation. VEGF siRNA also significantly inhibited E(2)-induced cell proliferation. In conclusion, E(2) partially stimulated hMSC proliferation via HIF-1alpha activation and VEGF expression through PKC, PI3K/Akt, and MAPK pathways.     

Effects of exogenous prostanoids on the proliferation of osteoblast-like cells in vitro

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.     

Short-period hypoxia increases mouse embryonic stem cell proliferation through cooperation of arachidonic acid and PI3K/Akt signalling pathways

Hypoxia plays important roles in some early stages of mammalian embryonic development and in various physiological functions. This study examined the effect of arachidonic acid on short-period hypoxia-induced regulation of G(1) phase cell-cycle progression and inter-relationships among possible signalling molecules in mouse embryonic stem cells. Hypoxia increased the level of hypoxia-inducible factor-1alpha (HIF-1alpha) expression and H2O2 generation in a time-dependent manner. In addition, hypoxia increased the levels of cell-cycle regulatory proteins (cyclin D(1), cyclin E, cyclin-dependent kinase 2 (CDK2) and CDK4). Maximum increases in the level of these proteins and retinoblastoma phosphorylation were observed after 12-24 h of exposure to hypoxic conditions, and then decreased. Alternatively, the level of the CDK inhibitors, p21(Cip1) and p27(Kip1) were decreased. These results were consistent with the results of [3H]-thymidine incorporation and cell counting. Hypoxia also increased the level of [3H]-arachidonic acid release and inhibition of cPLA(2) reduced hypoxia-induced increase in levels of the cell-cycle regulatory proteins and [3H]-thymidine incorporation. The level of cyclooxygenase-2 (COX-2) was also increased by hypoxia and inhibition of COX-2 decreased the levels of cell-cycle regulatory proteins and [3H]-thymidine incorporation. Indeed, the percentage of cells in S phase, levels of cell cycle regulatory proteins, and [3H]-thymidine incorporation were further increased in hypoxic conditions with arachidonic acid treatment compared to normoxic conditions. Hypoxia-induced Akt and mitogen-activated protein kinase (MAPK) phosphorylation was inhibited by vitamin C (antioxidant, 10(-3) M). In addition, hypoxia-induced increase of cell-cycle regulatory protein expression and [(3)H]-thymidine incorporation were attenuated by LY294002 (PI3K inhibitor, 10(-6) M), Akt inhibitor (10(-6) M), rapamycin (mTOR inhibitor, 10(-9) M), PD98059 (p44/42 inhibitor, 10(-5) M), and SB203580 (p38 MAPK inhibitor, 10(-6) M). Furthermore, hypoxia-induced increase of [(3)H]-arachidonic acid release was blocked by PD98059 or SB203580, but not by LY294002 or Akt inhibitor. In conclusion, arachidonic acid up-regulates short time-period hypoxia-induced G(1) phase cyclins D(1) and E, and CDK 2 and 4, in mouse embryonic stem cells through the cooperation of PI3K/Akt/mTOR, MAPK and cPLA(2)-mediated signal pathways.     

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-( >3 hr) and dose-(> 25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1-S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10(-6) M), Akt (Akt inhibitor, 10(-5) M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10(-5) M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [3H] arachidonic acid ([3H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 microCi of [3H] AA and with 200 micrograms radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [3H] AA to PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and to unidentified compounds in three chromatographic regions. One of these regions (region I) contained triglycerides. Endometrial slices metabolized only small amounts of the [3H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF2 alpha (32.1 +/- 17.9), PGFM (8.4 +/- 6.2), PGE2 (12.3 +/- 7.5) and 6-keto-PGF1 alpha (41.4 +/- 4.8), whereas day 16 conceptuses released more (P less than .05) PGF2 alpha (9.0 +/- 4.1) and 6-keto-PGF1 alpha (15.9 +/- 2.7) than PGE2 (0.9 +/- 0.2) or PGFM (0.5 +/- 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P less than .05) PGFM (3.0 +/- 0.2) and 6-keto-PGF1 alpha (4.0 +/- 0.4) than PGF2 alpha (0.5 +/- 0.08) or PGE2 (0.05 +/- 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [3H] AA to determine the effects of indomethacin on [3H] AA metabolism. In general, indomethacin (Id; 4 X 10(-4) M) reduced (P less than .05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [3H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [3H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embryonal survival and development, were synthesized in vitro by ovine conceptuses.     

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Effects of cortisol on serially propogated fibroblast cell cultures derived from the rabbit retal lung and skin.

Cortisol affects the growth of serially propogated, fibroblast cell cultures derived from the rabbit fetal lung in a manner which is dependent upon the gestational age of the material used: early in gestation (20 days), the hormone (10(-7)-10(-5) M) stimulates [6-3H]thymidine incorporation into DNA, while in late gestation (28 days), cortisol (10(-7) and 10(-6) M) inhibits this process. Cultures derived from the rabbit fetal skin are inhibited by cortisol (10(-5) M) at both gestational ages. Fibroblasts derived from lung, but not from skin, efficiently convert cortisone to cortisol and this activity increases with advancing gestation. Cortisol does not affect the incorporation of [3H]choline into lecithin by confluent cultures of any of the fibroblast types studied.     

Requirement for prostaglandin F2 alpha in 17 beta-estradiol stimulation of DNA synthesis in rabbit endometrial cultures

We have hypothesized that two of the endogenously synthesized endometrial prostaglandins (PGs), prostaglandin F2 alpha (PGF2 alpha), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17 beta-estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17 beta-estradiol stimulation resulted in a time dependent (optimal: approximately 6 h) and 17 beta-estradiol concentration dependent (optimal: approximately 10(-7) M 17 beta-estradiol in phenol red-containing medium) increase in [3H]PGF2 alpha binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2 alpha remained the same as in the control treated cultures. This 17 beta-estradiol stimulated increase in PGF2 alpha receptor allowed a suboptimal concentration of PGF2 alpha (10(-9) M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2 alpha caused no significant change in intracellular inositol polyphosphate levels. 17 beta-estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2 alpha binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17 beta-estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2 alpha was added to the medium at a concentration (10(-10) M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17 beta-estradiol stimulation, addition of these same low concentrations of PGF2 alpha had no effect on DNA synthesis. Apparently, through its effect on the PGF2 alpha receptor, 17 beta-estradiol enhances the PGF2 alpha stimulated DNA synthesis response approximately 100 fold. The DNA synthesis induced by 17 beta-estradiol can be inhibited by PGE1, as can PGF2 alpha-induced DNA synthesis. We propose that 17 beta-estradiol may be mediating its mitogenic effect through an alteration of the prostaglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In addition we suggest that, if 17 beta-estradiol acts to increase PGF2 alpha, receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17 beta-estradiol receptors.     

Prostaglandin F2 stimulates the generation of lipoxygenase products in guinea pig isolated trachea

The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.     

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Although the importance of estradiol-17 (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10^–9 M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10^–12 M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10^–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)- and - protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E₂-BSA; 10^–9 M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10^–12 M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10^–5 M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17 stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes. cyclin-dependent kinase; mitogen-activated protein kinase     

Influence of dexamethasone phosphate upon the DNA- and the NAD-metabolism of concanavaline a stimulated T-lymphocytes

1. Glucocorticoids have a decisive function in the immune system. In this paper, special attention is paid to the DNA and the NAD metabolism in T-lymphocytes of mice stimulated by Con A under the influence of dexamethasone phosphate. 2. Nicotinamide increases the incorporation of [3H]thymidine into the DNA of T-cells in dependence on the concentration. There is a similar but less pronounced effect with 1-methylnicotinamide. 3. Dexamethasone phosphate even at 10(-9) M inhibits the incorporation of [3H]thymidine into DNA. 4. The incorporation of [3H]thymidine into the DNA is reduced after preincubation of the T-cells with 6-aminonicotinamide or with 3-acetylpyridine. 5. Dexamethasone phosphate decreases the content of NAD in the T-cells. 6. The activity of the ADPR transferase increases after addition of Con A. Presence of nicotinamide stimulates the effect of Con A on this enzyme. This is not the case with 1-methylnicotinamide. The enzyme is inhibited drastically by dexamethasone phosphate. 7. It may be concluded that the NAD-adenoribosylation metabolism is markedly influenced by the mitogen Con A and by dexamethasone phosphate.     

Prostaglandin A1 induces differentiation in Friend erythroleukemia cells.

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B2 and 6-keto PGF1 alpha, were completely inactive, while PGE1 inhibited slightly and PGF2 alpha stimulated the replication of FLC. PGA1 was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA1 alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA1-stimulated differentiation was completely suppressed by the addition of 10(-6)M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA1 (but no DMSO) prevented the return to the undifferentiated state.