Stimulated Thymidine Uptake Research Articles

175. EFFECTS OF SPECIES DIFFERENCES ON OOCYTE REGULATION OF GRANULOSA CELL PROLIFERATION

The role of oocytes in regulating ovulation quota between species is not fully understood. In sheep, the oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have profound effects on ovarian follicular development and ovulation-quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled thymidine uptake by granulosa cells (GC) both within and between the two species. For these experiments, 32 oocytes denuded of cumulus-cells were co-incubated with 20 000 GC from either species. Rat or sheep oocytes stimulated thymidine uptake by GC from the same species (P < 0.005). Sheep oocytes also stimulated thymidine uptake by rat GC (P < 0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with 3.2 µg/mL or 7.6 mg/mL monoclonal antibodies specific to GDF9 or BMP15 respectively and to a hydatids antigen (control). Both sheep and rat oocyte stimulation of thymidine uptake by GC was inhibited with the GDF9 antibody (P < 0.05) but not control, irrespective of species of GC. Sheep oocyte stimulation of rat GC was also inhibited using an antibody to BMP15 (P < 0.05). However, when using the BMP15 antibody to block the effects of rat oocytes on rat GC, the inhibition of thymidine uptake was modest (i.e. ~10%) albeit significant (P < 0.05). The molecular forms of GDF9 and BMP15 in spent media were examined by Western blotting under reducing conditions. For both species, oocyte-secreted GDF9 was present in the mature form. For sheep oocytes, secreted BMP15 was present as promature and monomeric mature forms whereas from rats, trace amounts of mature form was sometimes, but not always, detected. Thus, in sheep both BMP15 and GDF9 are essential for regulating GC proliferation whereas in rats, although responsive to BMP15, GC proliferation is likely regulated primarily by GDF9.

Identification of Second Messenger Pathways Involved in the Cooperative Effects of Ovine GDF9 & BMP15 Stimulated Thymidine Uptake by Rat Granulosa Cells.

Identification of Second Messenger Pathways Involved in the Cooperative Effects of Ovine GDF9 & BMP15 Stimulated Thymidine Uptake by Rat Granulosa Cells.

272. Signalling pathways involved in mouse GDF9 and BMP15 stimulated thymidine uptake by rat granulosa cells

The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth and development. Understanding the molecular mechanisms of these factors could assist with the development of future products for fertility control. Thymidine uptake by rat granulosa cells is stimulated cooperatively by GDF9 and BMP15. Inhibitors of the activin receptor-like kinase (ALK) 4,5,7 and the nuclear factor kappaB (NFKB) second messenger pathways block ovine GDF9 and BMP15 stimulated thymidine incorporation. The ALK 4,5,7 receptor pathway is known to be essential for the cooperative effects of mouse (m)GDF9 and mBMP15 on thymidine incorporation but the role of other pathways has yet to be determined, which was the focus of this study. Inhibitors of NFKB (Sn50; 10µg/mL), ALK 2,3,6 receptor (Dorsomorphin; 1µM), p38 mitogen-activated protein kinase (p38 MAPK; SB239063; 5 µM) and c-Jun-N-terminal kinase (JNK; TAT-TI-JIP153–163; 5 µM) pathways were each cultured with recombinant mGDF9 (25 ng/mL) and mBMP15 (6 ng/mL) in a rat granulosa cell [3H]-thymidine bioassay. The p38 MAPK inhibitor caused partial inhibition of thymidine uptake but this appeared to be non-specific as a similar level of suppression was observed in the control cultures. Neither the ALK 2,3,6 receptor nor the NFKB pathway inhibitors had any effect on mGDF9 and mBMP15 stimulated thymidine uptake. The JNK inhibitor showed a 1.7-fold increase in stimulation above the mGDF9 and mBMP15 effect (P < 0.01) but a similar stimulation was also observed in some controls. This differs from the results observed with ovine GDF9 and BMP15 where thymidine uptake was completely blocked by the NFKB inhibitor and the JNK inhibitor had no effect. In conclusion, the molecular mechanisms of GDF9 and BMP15 function are dependent on the species of origin of the growth factor and therefore caution is needed when extrapolating findings from one species to another.

Nongenomic activity and subsequent c-fos induction by estrogen receptor ligands are not sufficient to promote deoxyribonucleic acid synthesis in human endometrial adenocarcinoma cells.

Estrogen 17beta-estradiol (E2) rapidly modulates several signaling pathways related to cell growth, preservation, and differentiation. The physiological role of these nongenomic effects with regard to downstream outcomes, and the relationship with transcriptional estrogen activity are unclear. Furthermore, the ability of selective estrogen receptor modulators (SERMs) to trigger nongenomic actions is largely unknown. To determine whether estrogen receptor (ER) ligands exert nongenomic activity in endometrial adenocarcinoma cells, and whether this activity affects transcription and DNA synthesis, we challenged human Ishikawa cells with E2 or partial ER agonists 4-hydroxytamoxifen (OHT) and raloxifene (ral). Serum-starved Ishikawa cells exposed for 5 min to 0.1 nM E2 showed induced phosphorylation of MAPK (ERK1/2). Ral and 4-OHT each at 1 nM also stimulated ERK in a rapid transient manner. E2 and 4-OHT induced proto-oncogene c-fos mRNA expression in Ishikawa cells within 30 min, but ral had no effect. In contrast to nongenomic action, only E2 stimulated expression of an estrogen response element (ERE)-driven luciferase (LUC) reporter gene. To examine DNA synthesis, [(3)H]-thymidine incorporation was measured in serum-starved cultures exposed to E2 or partial agonists for 2 d. E2 at 1 nM stimulated thymidine uptake in an ERK-dependent manner, but 1 nM 4-OHT, 1 nM ral, and 0.1-nM concentrations of E2 had no significant effects. Taken together, these data indicate that both nongenomic and direct transcriptional ER effects are likely required to promote DNA synthesis.

Extracellular signal-regulated kinases and g protein-coupled receptors in megakaryocytic human erythroleukemia cells: selective activation, differential regulation, and dissociation from mitogenesis.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are a group of kinases that play an important role in proliferation and differentiation. In megakaryocyte-like human erythroleukemia (HEL) cells, ERK2 was found to be predominantly expressed and strongly activated by prostaglandin (PG) E(2), thrombin, and epinephrine. On the other hand, adenosine, ADP, ATP, and UTP did not significantly increase ERK1/2 phosphorylation. However, of the agonists tested, only ADP was able to stimulate thymidine uptake. Pretreatment with pertussis toxin abolished the PGE(2) response but had less of an effect on thrombin. PGE(2)- and thrombin-induced ERK1/2 activation was mimicked by 4-beta-phorbol-12-myristate-13-acetate and ionomycin and blocked by mitogen-activated protein kinase kinase inhibitor 1,4 diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene but displayed differential sensitivity to protein kinase C inhibitor bisindolylmaleimide I and Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Analogs of cAMP or agents that stimulate cAMP production were either weak or ineffective activators. Further studies indicate that the effect of thrombin was blocked by the phosphoinositide 3-kinase inhibitor wortmannin but not by agents inhibiting tyrosine kinase activity. On the contrary, herbimycin, but not wortmannin, attenuated the effect of PGE(2). Collectively, these results indicate that ERK1/2 are selectively activated by G protein-coupled receptors and not functionally associated with proliferation in HEL cells. ERK1/2 activation in response to PGE(2) and thrombin is mediated by distinctive types of G proteins and is differentially regulated by multiple pathways, including calcium mobilization, protein kinase C, phosphoinositide 3-kinase, and tyrosine kinases.

Mast cells express functional CD30 ligand and are the predominant CD30L-positive cells in Hodgkin's disease.

Hodgkin's disease (HD) tumours are characterized by the presence of few tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, surrounded by a large amount of non-neoplastic cells. The role of this cell infiltrate for the development of HD is not known. CD30, belonging to the tumour necrosis factor receptor superfamily, is highly expressed on HRS cells and believed to be involved in tumourigenesis and tumour progression. Tumour samples from 42 patients were immunohistochemically double-stained for tryptase, a mast cell-specific proteinase and CD30 ligand (CD30L). Tryptase-positive mast cells were present in all tumours. Of these cells, 50% expressed CD30L and 66% of the CD30L-positive cells were mast cells. CD30L mRNA in in vitro developed normal mast cells and malignant human and murine mast cell lines was detected using reverse transcription polymerase chain reaction. CD30L protein expressed on human mast cells was detected using flow cytometry. In a co-culture assay, the human mast cell line HMC-1 stimulated thymidine uptake in HRS cell lines, and the stimulation could be blocked using CD30L-specific monoclonal antibodies. In conclusion, mast cells are present in HD tumours and are the predominant CD30L-expressing cells. CD30L-CD30 interaction is a pathway by which mast cells may stimulate DNA synthesis in HRS cells.

Proliferation of rat pleural mesothelial cells in response to hepatocyte and keratinocyte growth factors.

The proliferative response of cultured pulmonary mesothelial cells (MCs) to epithelial cell mitogens such as keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) is investigated. A cell line of rat pleural MCs and freshly prepared rat visceral and parietal MCs were studied. Both KGF and HGF stimulated thymidine uptake in the cell line when cultured for 2 d in serum-free conditions; the growth increase was magnified when tumor necrosis factor (TNF)-alpha was also added to the cultures. Adding asbestos fibers alone to MCs in culture did not enhance DNA synthesis by these cells. The MCs were also shown to synthesize significant amounts of HGF but much less KGF when cultured for 2 d. When freshly prepared MCs were examined, normal cell growth was more rapid in the parietal cells, which also had a more epithelial-type morphology. The addition of HGF and KGF resulted in increased DNA synthesis in each cell type, but no effect of added TNF-alpha was found. The results indicate that pulmonary MCs have the potential to proliferate in response to cytokines such as HGF and KGF that are usually associated with epithelial cell regeneration after injury.

Suppression of tyrosine kinase activity inhibits [3H]thymidine uptake in cultured human pituitary tumor cells.

Tyrosine kinases are involved in the phosphorylation of proteins that regulate cell growth and proliferation. The mitogenic effect of several growth factors requires tyrosine kinase activity of their receptors. The effect of inhibition of tyrosine kinase activity on thymidine uptake into cultured human pituitary adenoma cells was studied using two inhibitors, genestein and methyl-2,3-dihydroxycinnamate (MDHC). Of 33 pituitary adenomas, 7 incorporated sufficient [3H]thymidine to be investigated in the experiments. Genestein and MDHC both potently inhibited thymidine uptake into these tumors, with a mean inhibition by 74 mumol/L genestein of 61.96 +/- 18.96% (+/- SD inhibition of basal), by 740 mumol/L genestein of 92.65 +/- 8.59%, and by 100 mumol/L MDHC of 93.84 +/- 3.85%. The 7 pituitary adenomas were all large with suprasellar extension and secreted interleukin-6 in vitro. They included 2 prolactinomas, 1 somatotropinoma, 1 mammosomatropinoma, and 3 clinically nonfunctioning adenomas. Epidermal growth factor stimulated thymidine uptake in 2 of the 3 clinically nonfunctioning adenomas studied, and this stimulation was inhibited by genestein. Both of these tumors released FSH in cell culture and are probably silent gonadotropinomas. The growth stimulatory effect of conditioned medium from human pituitary cell culture on GH3 cells was inhibited by both genestein and MDHC. We conclude that tyrosine kinase activity is crucial for the integrity and growth of pituitary adenomas in culture. Growth factors released by pituitary adenomas potentially may maintain and promote tumor growth by stimulating tyrosine kinase activity.

Growth hormone stimulates the proliferation of activated mouse T lymphocytes.

A modulatory role for GH on immune function has been suggested, but hormonal effects have been difficult to demonstrate with isolated cells. We have recently shown that GH receptors are present in murine hematopoietic tissues, with a lower receptor number in T lymphocytes than in B cells or macrophages. The binding of bovine GH (bGH) to murine splenocytes is increased after T cell activation with either concanavalin A or anti-CD3 antibody. In the present study, we show that bGH is able to stimulate the proliferation of activated murine T cells. Splenocytes were stimulated with either Con A or anti-CD3 antibody; addition of the mitogen resulted in increased [3H]thymidine uptake. When added together with the mitogen to the culture medium, bGH was able to further stimulate thymidine uptake. A bell-shaped dose-response curve was observed. bGH was able to increase cell proliferation by 2.5-fold over the effect of anti-CD3 alone. The amplitude of the bGH response was greater in unfractionated splenocytes than in purified T lymphocytes or thymocytes. Splenocytes were also stimulated by lipopolysaccharide, a B cell-specific mitogen; no change in the level of bGH binding was observed during activation of B cells, and no effect of bGH on the proliferative response of splenocytes to lipopolysaccharide was detected. The GH proliferative effect on T lymphocytes is probably direct and not through locally produced insulin-like growth factor I, because insulin-like growth factor I did not affect the cell proliferation when added at concentrations ranging from 10(-9)-10(-7) M. Ovine PRL was also able to stimulate [3H]thymidine uptake in splenocytes and thymocytes, and a synergistic effect was observed when bGH and ovine PRL were added together at 10(-8) M. Our findings support the biological significance of the GH receptors identified in murine T lymphocytes and confirm the role of GH in the regulation of immune functions.

Activation of the Erythropoietin (EPO) Receptor by Bivalent Anti-EPO Receptor Antibodies

Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34(+) cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose.

Antitumor effects of liposomal IL1? and TNF? against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1 alpha) and tumor necrosis factor alpha (TNF alpha) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37 degrees C. The biological activities mediated by liposomal IL1 alpha and TNF alpha were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF alpha-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 alpha and TNF alpha significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 alpha and TNF alpha displayed significant in vivo antitumor activity against the IL1 alpha- and TNF alpha-resistant B16F10 metastatic murine melanoma.

Vasopressin and vasopressin-receptor immunoreactivity in small-cell lung carcinoma (SCCL) cell lines: disruption in the activation cascade of V 1a-receptors in variant SCCL

Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V 1a-receptor immunoreactivity. One of these classical cell lines, NCI-H345, and one variant cell line, NCI-H82, were further investigated for binding of V 1 and V 2 vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCI-H345 demonstrated similar binding patterns with the V 1 and V 2 vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 μM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCI-H82). However, vasopressin (0.1 or 1 μM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V 1a and V 2 receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V 1a receptors as indicated by the lack of vasopressin-induced calcium mobilization.

Parathyroid hormone-related protein: A potential autocrine growth regulator in human prostate cancer cell lines

Objective. We recently demonstrated that parathyroid hormone-related protein (PTHrP) is widely expressed by human prostate cancer tissue, suggesting that PTHrP might be involved in the growth and development of prostate cancer. To study this further, the production of PTHrP and its biologic effect were investigated using human prostate cancer cell lines. Methods. The cell lines used were one androgen-dependent cell line, LNCaP, and two androgen-independent cell lines, PC-3 and DU-145. PTHrP secreted by cancer cells was measured by radioimmunoassay. The effect of PTHrP on DNA synthesis in these cells was determined by thymidine incorporation assay. Results. All cell lines secreted immunodetectable levels of PTHrP in the culture-conditioned media. PC-3 cells secreted significantly higher amounts than the other two cell lines. A synthetic peptide, PTHrP(1–34), stimulated thymidine uptake in PC-3 and DU-145 cells more than threefold the control under serum-free and steroid-free conditions, whereas LNCaP was not affected. However, in the presence of dihydrotestosterone, DNA synthesis of LNCaP cells was stimulated by PTHrP in a dose-dependent manner. Additionally, this PTHrP-induced DNA synthesis was completely neutralized by a validated mouse monoclonal antibody (8131 2) raised against PTHrP(1–34). Conclusions. Our data suggest that PTHrP may play a significant role in the growth of prostate cancer by acting locally in an autocrine fashion.

Activated murine neutrophils induce unscheduled DNA synthesis in B lymphocytes

Activated neutrophils induce DNA damage in neighboring cells by secreting reactive oxygen compounds into the extracellular milieu. Repair of this damage is required to prevent mutagenesis and neoplastic transformation. Conditions were established to detect the activation of excision-repair pathways (unscheduled DNA synthesis) by measuring stimulated thymidine uptake in target B lymphocytes exposed to activated neutrophils. Murine neutrophils were cocultured in serum-free medium with splenic B cells or with murine plasmacytoma cells for 2 h. Unscheduled DNA synthesis in the B cells was detected at neutophil: target cell ratios of 1:1 to 4:1 when the neutrophils were activated with phorbol myristate acetate. Reagent H2O2 alone (≥6 μM) also induced UDS whereas HOCl (up to 4 mM) did not. No repair synthesis was observed within the neutrophils themselves. Control experiments indicated that the induction of UDS by neutrophils and H2O2 was not due to formation of a stable genotoxic compound from HU. On the contrary, scavenging of free H2O2 by HU probably lowered the levels of UDS that could be detected by these agents. Induction of unscheduled DNA synthesis by neutrophils and H2O2 occurred under conditions of less cytostasis than was found with other DNA-damaging agents such as 4-nitroquinoline 1-oxide or γ-irradiation. This may reflect a heightened responsiveness of the cells to repair of damage from physiological oxidants. The result demonstrate that DNA damage induced by reactive oxygen intermediates can be repaired by nucleotide excision-repair pathways.

Hormonal regulation of the growth and steroidogenic function of human granulosa cells

The aim of this study was to assess development-related interactions between gonadotropins and insulin-like growth factor (IGF-I) on DNA synthesis and steroidogenesis in human granulosa cells. "Immature" granulosa cells were obtained from follicles during the late luteal phase or first half of the follicular phase; "mature" granulosa cells came from follicles during the second half of the follicular phase but before the midcycle LH surge; and granulosa-lutein cells were obtained as a by-product of in vitro fertilization. Granulosa cells were cultured for 96 h in serum-free medium 199 with and without LH or FSH, and in the presence and absence of IGF-I. The cell monolayers were then incubated with [3H]methyl thymidine to assess DNA synthesis. Spent culture medium was assayed for progesterone and estradiol content. Immature granulosa cells: Tritiated thymidine uptake in granulosa cell cultures from immature follicles were significantly increased by IGF-I. FSH was able to maintain or increase basal and IGF-I stimulated growth whereas LH had no effect. Basal progesterone production was low and not increased by either FSH or LH. However, treatment with FSH, but not LH, increased aromatase activity. Mature granulosa cells: IGF-I also stimulated thymidine uptake. However, whereas FSH either maintained or increased thymidine uptake by these cells, LH dose dependently suppressed thymidine uptake. This inhibitory action of LH was accentuated by the presence of IGF-I. Despite the inhibitory effect of LH on thymidine uptake, the gonadotropin markedly stimulated steroid production and the maximal steroidogenic response to LH was equivalent to 3-fold greater than that to FSH. Granulosa-lutein cells: Patterns of basal and IGF-I- and gonadotropin-stimulated steroid synthesis were similar to those observed for mature granulosa cells but steroid production rates were higher. Suppression of basal and IGF-I-stimulated thymidine uptake by LH was even more pronounced. These results suggest that the granulosa cell LH receptor, once expressed, negatively regulates cell growth and, simultaneously, positively regulates steroid synthesis. This development related event could be crucial to the mechanism whereby granulosa cells cease to divide and commence maximal rates of steroid synthesis in response to the LH surge.

Hormonal regulation of the growth and steroidogenic function of human granulosa cells.

The aim of this study was to assess development-related interactions between gonadotropins and insulin-like growth factor (IGF-I) on DNA synthesis and steroidogenesis in human granulosa cells. "Immature" granulosa cells were obtained from follicles during the late luteal phase or first half of the follicular phase; "mature" granulosa cells came from follicles during the second half of the follicular phase but before the midcycle LH surge; and granulosa-lutein cells were obtained as a by-product of in vitro fertilization. Granulosa cells were cultured for 96 h in serum-free medium 199 with and without LH or FSH, and in the presence and absence of IGF-I. The cell monolayers were then incubated with [3H]methyl thymidine to assess DNA synthesis. Spent culture medium was assayed for progesterone and estradiol content. Immature granulosa cells: Tritiated thymidine uptake in granulosa cell cultures from immature follicles were significantly increased by IGF-I. FSH was able to maintain or increase basal and IGF-I stimulated growth whereas LH had no effect. Basal progesterone production was low and not increased by either FSH or LH. However, treatment with FSH, but not LH, increased aromatase activity. Mature granulosa cells: IGF-I also stimulated thymidine uptake. However, whereas FSH either maintained or increased thymidine uptake by these cells, LH dose dependently suppressed thymidine uptake. This inhibitory action of LH was accentuated by the presence of IGF-I. Despite the inhibitory effect of LH on thymidine uptake, the gonadotropin markedly stimulated steroid production and the maximal steroidogenic response to LH was equivalent to 3-fold greater than that to FSH. Granulosa-lutein cells: Patterns of basal and IGF-I- and gonadotropin-stimulated steroid synthesis were similar to those observed for mature granulosa cells but steroid production rates were higher. Suppression of basal and IGF-I-stimulated thymidine uptake by LH was even more pronounced. These results suggest that the granulosa cell LH receptor, once expressed, negatively regulates cell growth and, simultaneously, positively regulates steroid synthesis. This development related event could be crucial to the mechanism whereby granulosa cells cease to divide and commence maximal rates of steroid synthesis in response to the LH surge.

Balb/C mice immunized with either xenogeneic or syngeneic TSH produce immunoglobulins with anti-TSH and thyroid-stimulating activities

Balb/C mice were immunized with bTSH (xenogeneic TSH) or extracts of Balb/C pituitaries (containing syngeneic TSH), either intracutaneously with Freund's adjuvant or intrasplenically. After bTSH, all blood samples contained anti-TSH antibodies. Thyroid-stimulating activity was assayed on FRTL-5 cells. Of 80 sera from immunized mice, 33 induced an increase in 99mTcO4 uptake, and 31/79 induced an increase in thyroidal 3[H]thymidine uptake by thyrocytes. With syngeneic TSH, anti-TSH antibodies were found in 10/19 mice. Immunoglobulins increasing thyrocyte technetium uptake were found in 14/19 mice, and immunoglobulins that stimulated thymidine uptake in thyrocyte DNA were detected in 12/19 sera. After immunization with both types of TSH, sera of some mice showed only one of the two bioactivities. Hybridomas were prepared with splenic lymphocytes of the mice immunized with either of the TSH preparations. These secreted TSH-binding immunoglobulins or immunoglobulins which stimulated thymidine or technetium uptake by the thyrocytes. One of the hybridomas from mice immunized with pituitary extract secreted a monoclonal antibody which stimulated thyroidal thymidine uptake, but inhibited bTSH-induced 99mTcO4 uptake. These findings might suggest that the anti-idiotypic network acting on TSH as an antigen could be involved in the pathogenesis of stimulatory antibodies in thyroid disease.

Mutational analysis of leucine 47 in human epidermal growth factor

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.

ラット培養メサンギウム細胞におけるInsulin-Like Growth Factor-I(IGF-I)作用と高糖濃度の影響

Recent studies indicate the important roles of mesangial cell dysfunction and insulin-like growth factor I (IGF-I) in the development of diabetic nephropathy. In order to know whether hyperglycemia could alter IGF-I action on mesangial cells, we examined mitogenic and metabolic effects of IGF-I on mesangial cells. Mesangial cells revealed to express considerable numbers of receptors specific to IGF-I will relatively small numbers of insulin receptors. The uptake of [3H]-2-deoxy-glucose, [3H]-aminoisobutyric acid (AIB), or [3H]-thymidine into mesangial cells was stimulated by IGF-I at physiological concentrations. Under high concentrations of glucose (55 mM), the stimulation of thymidine uptake by IGF-I was significantly suppressed from 5863 +/- 549 (at 11 mM glucose) to 1731 +/- 146 DPM/100 micrograms/prot. On the contrary, AIB incorporation by IGF-I was significantly enhanced in the cells cultured under high concentration of glucose, as 2.03 +/- 0.03n mol/mg protein/15 min at 55 mM glucose vs 0.59 +/- 0.01 at 11 mM glucose. In conclusion; 1) IGF-I had metabolic and mitogenic effects on rat mesangial cells at physiological concentrations. 2) under excess glucose conditions, mitogenic action of IGF-I on rat mesangial cells was suppressed, while amino acid incorporation was enhanced. These results suggest that modulation of IGF-I effects on mesangial cell by glucose could be associated with mesangial cell dysfunction in diabetes.

Fluoride and Bovine Bone Extract Influence Cell Proliferation and Phosphatase Activities in Human Bone Cell Cultures

The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of factors. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.