Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.     

Identification of neutrophil alkaline phosphatase-inducing factor in cystic fluid of a human squamous cell carcinoma as granulocyte colony-stimulating factor

We have previously shown that a factor termed neutrophil alkaline phosphatase-inducing factor (NAP-IF) has the capacity to induce neutrophil alkaline phosphatase (NAP) in postmitotic granulocytes (PMGs). This factor has characteristics similar to those of granulocyte colony-stimulating factor (G-CSF), suggesting that the two factors assayed by different methods may be attributable to an identical macromolecule. In a preliminary experiment, we showed that purified natural G-CSF (nG-CSF) could induce NAP in vitro in the presence of 10% (v/v) fetal calf serum (FCS). In this study, purified human nG-CSF and recombinant G-CSF (rG-CSF) induced NAP in granulocytes from both normal individuals and patients with chronic myelogenous leukemia in a dose-dependent fashion in serum-free and serum-containing culture conditions. The induction of NAP by G-CSF was detectable at 0.4 ng/ml and became maximal between 10 and 20 ng/ml. Anti-G-CSF serum incubated with either NAP-IF or rG-CSF inhibited induction of NAP. Morphological examinations revealed that granulocytes cultured with G-CSF were more mature than those cultured without G-CSF, indicating that G-CSF promoted maturation of granulocytes in parallel with NAP induction. These results indicate that NAP-IF in the cystic fluid of a human squamous cell carcinoma is identical to G-CSF and that induction of NAP by G-CSF is really a reflection of cell maturation promoted by G-CSF.     

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.     

Cyclooxygenase-2 activity is necessary for the angiogenic properties of oncostatin M.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)-6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC-1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC-1, OSM (0.5-2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL-1beta (5 ng/ml) induced production of cyclooxygenase (COX)-2. In contrast, in HUVECs, neither OSM nor LIF induced COX-2 mRNA, suggesting that COX-2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM-induced HMEC-1 proliferation of specific COX-2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX-2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL-1beta, which also induces COX-2 in HMEC-1, has only a poor proliferative effect.     

Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.     

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.     

Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins.

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.     

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Gonadotrophic control of steroidogenesis in human granulosa-lutein cells

Granulosa-lutein cells were harvested from periovulatory follicles in human ovaries and cultured for up to 6 days, equivalent to almost half of a normal luteal phase. The average rate of basal progesterone accumulation in the culture medium was constant at approximately 36 nmol progesterone/10(6) cells/day. Oestradiol accumulation was too low to measure in the absence of precursor androgen. Basal aromatase activity (measured as oestradiol formed in 3 h from 10(-6) M exogenous testosterone) was high (average 1.15 nmol oestradiol/10(6) cells/3 h) at the time of cell isolation (Day 0) but fell by greater than 90% on Day 1. By Day 2 the activity had partly recovered and averaged 62% of the Day 0 value, rising to 70% on Day 6. This loss and recovery of aromatase activity was independent of the addition of gonadotrophic hormones to the culture medium. However, dose-related increases in aromatase activity occurred in the presence of highly pure human pituitary LH (0.1-30 ng/ml). The increase was observed on Day 4 and was maximal on Day 6 (average 3-fold increase over control) in the presence of LH concentrations greater than or equal to 1.0 ng/ml. LH also caused dose-related increases in progesterone accumulation by Day 4 with maximal stimulation on Day 6 (average 3-fold increase over control) at greater than or equal to 10.0 ng/ml. Dose-related stimulation of aromatase activity by human pituitary FSH also occurred but maximal stimulation required the presence of 300 ng FSH/ml and progesterone accumulation was hardly affected at this dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells.

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone.

The effect of different doses of luteinizing hormone on activation of protein kinases, cyclic AMP and testosterone production was studied in purified rat testis Leydig-cell preparations in the presence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). In addition, the nature of the protein kinases present in these cells and other tissues was investigated. The following results were obtained. 1. With all the amounts of luteinizing hormone used (0.1-1000 ng/ml), both activation of protein kinase and stimulation of testosterone production were demonstrated. With the lowest amount of luteinizing hormone (0.1 ng/ml), an 8.4+/-0.9% (S.E.M.,n=6) stimulation of protein kinase activation occurred, increasing to 100% with 1000 ng/ml, compared with 3.2+/-1.0%(S.E.M.,n=7) and 100% stimulation of testosterone production with 0.1 and 100 ng/ml respectively. 2. With amounts of luteinizing hormone up to 1 ng/ml (which gave half-maximal stimulation of testosterone production) no detectable increases in net cyclic AMP production were obtained. With higher amounts of luteinizing hormone, cyclic AMP production increased, but maximal production was not reached with 1000 ng/ml. 3. Two isoenzymic forms of protein kinase were present in Leydig cells and seminiferous tubules; type I was eluted with 0.075 M-and type II with 0.22-0.25 m-NaCl from DEAE-cellulose columns. 4. The protein kinase activity was not affected by the presence of erythrocytes in the Leydig-cell preparation, but varied depending on the type of histone used as substrate (histone F2b greater than mixed greater than histone F1).     

Formation of estrogen glucuronides by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization

The formation of steroid glucuronides by human granulosa cells isolated from human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG)-stimulated cycles for in vitro fertilization was studied. From granulosa cells in suspension, 5 x 10(-7) M androstenedione was converted into estradiol (2.50 +/- 0.21 ng/ml), estrone (1.84 +/- 0.16 ng/ml), estradiol glucuronide (0.38 +/- 0.07 ng/ml), as well as estrone glucuronide (0.24 +/- 0.04 ng/ml). When 5 x 10(-7) M estradiol was incubated, estrone (15.5 +/- 0.9 ng/ml) and estradiol glucuronide (0.12 +/- 0.05 ng/ml) were detected in medium. Using the same preparation of granulosa cells, we have observed that androsterone could uniquely be transformed into androstane-3 alpha, 17 beta-diol (1.42 +/- 0.56 ng/ml), and only low amounts of steroid glucuronides could be detected. Since the formation of steroid glucuronides was extremely small when granulosa cells in suspension were used, we subsequently studied granulosa cells in culture. When 5 x 10(-7) M estradiol was added, estrone (7.8 +/- 1.3 ng/ml) and estradiol glucuronide (0.68 +/- 0.08 ng/ml) were formed. The addition of follicle-stimulating hormone did not cause a further increase in estrone or estradiol glucuronide levels. As observed with granulosa cells in suspension, incubation with androsterone led to the formation of androstane-3 alpha, 17 beta-diol (24.2 +/- 0.07 ng/ml). Our data demonstrated the presence of glucuronyltransferase in human granulosa cells obtained from preovulatory follicles of hMG/hCG-treated women. In addition, since the conversion of androsterone into C-19 steroid glucoronide was relatively small, the present finding also indicates that the glucoronyltransferase enzymatic activity in granulosa-luteal cells preferentially conjugated estrogens.     

Granulocyte colony-stimulating factor potentiates anti-Candida albicans growth inhibitory activity of polymorphonuclear cells

Abstract Granulocyte colony-stimulating factor (G-CSF) stimulates a subset of granulocyte colony forming cells and when administered to neutropenic individuals results in recovery of blood neutrophil numbers to normal levels. Therefore, G-CSF may be a useful therapeutic agent for infections in immunocompromised hosts. However, to date there has been only limited information that G-CSF activates the antimicrobial activity of neutrophils. In the present study, we found that recombinant G-CSF promotes the anti- Candida albicans activity of normal human blood polymorphonuclear (PMN) cells in vitro using both a ³H-glucose uptake procedure and a Candida colony counting assay. As little as 0.1 ng/ml G-CSF induced significant anti-Candida activity in the PMN cultures. G-CSF treatment also enhanced superoxide anion production by the PMNs in response to f-MLP as determined by the superoxide dismutase inhibitable cytochrome C reduction method. Such results show that G-CSF can promote the antimicrobial activity of peripheral blood PMNs against C. albicans .     

Biological activity of the seminal plasma of alpacas: stimulus for the production of LH by pituitary cells

South American camelids are induced ovulators and require a stimulus to trigger the LH surge responsible for the ovulation. Seminal plasma (SP) of fertile alpacas (Lama pacos) was tested using a bioassay of pituitary cells to study the effect of seminal plasma on LH release. Plates containing rat pituitary cells (2 x 10(5) cells/90-95% viability) were cultured adding: (A) whole SP (WSP) treated with charcoal-dextran, or 1:2 or 1:4 proportions diluted in culture medium (DMEM/HEPES + antibiotics), or (B) 1:2 SP + anti-GnRH rabbit serum (inhibitory potency 10(-5) M), or (C) 1:2 SP + anti-GnRH + 100 nM synthetic GnRH (buserelin acetate) or (D) 100 nM, 50 nM, 10 nM, and 1 nM synthetic GnRH. Concentration (ng/ml) of LH secreted (Sec) and contained (Con) was analyzed using RIA 125I and the percentage of Sec and Con in each experiment was determined. The results of LH Sec for the cells treated with 50, 10, and 1 nM GnRH were 39, 13, and 1.5%, respectively (r2 = 98.41%, r = 0.9920) but cells treated with 100 nM GnRH secreted 10% of LH. With WSP, 1:2, or 1:4 SP the LH Sec was of 44.5% (3.25 ng/ml), 27% (1.9 ng/ml), and 18% (1.2 ng/ml), respectively. The exposure of cells to 1:2 SP + anti-GnRH, or to 1:2 SP + anti-GnRH/100 nM GnRH produced 31% (2.20 ng/ml) and 30% (1.8 ng/ml) of LH Sec, respectively. These results suggest that the SP of alpacas could have some factor(s) different from GnRH that would contribute to the mechanisms of LH secretion and to the induced ovulation in the female alpaca.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

Type beta transforming growth factor (TGF beta) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells

TGF beta 1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three-fold. This effect was dose-dependent with an ED50 of about approximately 0.2 ng/ml and was larger during logarithmic growth than at confluence. TGF beta 1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGF beta 1 was sufficient to increase AP activity 3 days later by about two-fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGF beta 1 addition and was blocked by cycloheximide and actinomycin D. TGF beta 1 also increased AP mRNA by two- to three-fold and this effect was not blocked by cycloheximide. The half-life of AP mRNA, estimated following the addition of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was about ten h in both control and TGF beta 1-treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGF beta 1 but fibronectin mRNA was decreased. TGF beta 2 effects on AP and cell growth were similar to those of TGF beta 1, except for lack of activity following transient exposure. At saturating concentrations, TGF beta 2 (2 ng/ml) or dexamethasone (10(-7) M), which has similar effects on these cells, did not further augment the effects of TGF beta 1 (at 2 ng/ml). Above findings suggest that TGF beta promotes osteoblastic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.     

Low and High Concentrations of the Topo II Inhibitor Daunorubicin in NIH3T3 Cells: Reversible G2/M Versus Irreversible G1 and S Arrest

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DNA. In both drug-sensitive 3T3 cells, and drug-resistant 3T3 cells, NIH-MDR-6815, (created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells), cells initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells and accumulated in G2/M. The 2 to 3 h lag period represents the time taken for fully establishing the G2/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G1 and S, and did not reach G2/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G2/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).     

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: change in apoptosis-related gene expression

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.