Term Monolayer Cultures Research Articles

A Role for Prolyl 3-Hydroxylase 2 in Post-translational Modification of Fibril-forming Collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.

Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.

Effect of gonadotropins on brain-derived neurotrophic factor secretion by human follicular cumulus cells

Effect of gonadotropins on brain-derived neurotrophic factor secretion by human follicular cumulus cells

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2.

Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2 to positively regulate chondrogensis.

Glucocorticoids potentiate the adenylyl cyclase—cAMP system mediated immunoreactive β-endorphin production and secretion from hypothalamic neurons in culture

Beta-endorphin(βEP) 1−31, a potent opioid peptide of proopiomelanocortin (POMC) derivatives, is produced and released from neurons at arcuate nuclei of the rat hypothalamus. Although dexamethasone (DM) suppresses the production and secretion of POMC related peptides from rat pituitary corticotrophs, the effect of glucocorticoids on the function of hypothalamic βEP neurons remains unclear. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 day treatment with 10 μM of forskolin increased ir-βEP levels in cell content and culture media by approximately1.7 ( P < 0.05) and 4.1 times ( P < 0.01) above vehicle treated control cultures (mean ±S.E.M., 47.3 ± 2.6 pg/well 40.4 ± 3.0 pg/well; n = 3) respectively. Although 4 day treatment with DM alone had little effect on the release and the cell content of ir-βEP, it significantly enhanced forskolin-induced elevation of ir-βEP levels in cell content and in culture media. The effect of DM was dose-related and time-dependent, with an EC 50 of about 1 nM; at this concentration DM enhanced ir-βEP secretion about2.1 times ( P < 0.01) above that induced by 10 μM of forskolin alone. Furthermore, the potentiating effect of DM was specifically suppressed by 100 nM of RU38486 ( P < 0.01), a glucocorticoid receptor antagonist, but not by an equivalent dose of RU28318, a mineralocorticoid receptor antagonist. In addition, Northern blot analysis showed that forskolin (10 μM) increased the abundance of POMC mRNA 1.4 fold above that of vehicle treated control cultures. Whereas by itself, DM (10 nM) had little effect on the level of POMC mRNA, it enhanced forskolin-stimulated increase of the abundance of POMC mRNA approximately 2.6 times. Moreover, DM also augmented1.6 times ( P < 0.05) forskolin-induced but not 3-isobutyl-1-methylxanthine (IBMX)-induced increase of cAMP production (5.5 ± 0.4 pmol/well; mean ± S.E.M., n = 3) in the cultures. Taken together, our findings suggest that in contrast to the inhibitory effect on pituitary corticotrophs, glucocorticoids enhance the production and secretion of βEP from rat hypothalamic neurons by facilitating the stimulatory effect mediated, in part, through the adenylyl cyclase-cAMP system.

Evaluation of separation and long term monolayer cultures of porcine hepatocytes for liver assist support . Dept. of Pathology and Lab Med, Rhode Island Hospital and Brown University, Providence, RI

Evaluation of separation and long term monolayer cultures of porcine hepatocytes for liver assist support . Dept. of Pathology and Lab Med, Rhode Island Hospital and Brown University, Providence, RI

Interleukin-1 beta stimulates colony-stimulating factor-1 production in placental villous core mesenchymal cells.

The human placenta is known to produce hematopoietic growth factors, including colony-stimulating factor-1 (CSF-1). We have previously demonstrated interleukin-1 beta (IL-1 beta) production by decidualized endometrium during pregnancy. Since IL-1 stimulates CSF-1 production in fibroblasts, endothelial cells, and bone marrow stromal cells, our present study was designed to determine whether IL-1 could also regulate CSF-1 production by placental villous core mesenchymal cells. Initial studies using enzymatic digestion separation of the trophoblast and villous core demonstrated that CSF-1 mRNA is mainly expressed in the placental villous core. Subsequently, long term monolayer cultures of villous core mesenchymal cells isolated from 9- to 16-week gestation placentas were established after enzymatic dissociation of intact placental villi to study the regulation of villous core cell CSF-1 production. The morphology of the cells and immunohistochemical staining for vimentin, cytokeratin, and CD45 antigen confirmed that cultured cells were more than 95% mesenchymal fibroblasts. Time-course experiments demonstrated a maximal increase in CSF-1 mRNA expression of approximately 6.0-fold over baseline levels 3 h after IL-1 beta treatment (10 ng/mL), whereas increased CSF-1 protein production was first detected in the culture medium 3 h after IL-1 beta treatment and rose progressively over 42 hours. Villous core mesenchymal cells incubated with 0-10 ng/mL IL-1 beta demonstrated a specific dose-response relationship for CSF-1 mRNA expression and protein production, first seen at 0.10 ng/mL and maximal with 10 ng/mL IL-1 beta. These results demonstrate that production of CSF-1 by placental villous core mesenchymal cells can be stimulated by IL-1 beta in vitro and suggest that decidual IL-1 may regulate placental CSF-1 production in vivo.

In vitro evidence for modulation of morphological and functional development of hypothalamic immunoreactive atrial natriuretic peptide neurons by cyclic 3',5'-adenosine monophosphate.

In the hypothalamus of the rat, the precursor of atrial natriuretic peptide (ANP) is produced and processed into its smaller congeners of 3K mol wt species, which are secreted from neurons with cell bodies in the periventricular areas and the paraventricular nuclei of the tissue. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we have identified a small population of cells that stained positive for immunoreactive (ir) ANP. Seventy-two +/- 7% (mean +/- SE; n = 4 per 1000 cells) of the irANP positive cells were colocalized with the staining of neuron-specific enolase; some of the cells possessed multiple neurites and showed irANP staining in the perikarya, in the varicosities along neuronal processes, and at the terminals of long neurites. Over the range of 10(-6)-10(-4) M, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP significantly augmented the total number of irANP-positive cells and those possessing neurites in a dose-related and time-dependent manner. At 10(-4) M, 4 days of forskolin treatment increased the number of irANP-positive neurons 4-fold (P less than 0.01) while tripling that of the cells with long neurites (P less than 0.01). Furthermore, it approximately tripled the number of cells (P less than 0.01) showing positive signals for pro-ANP mRNA, as ascertained by colorimetric in situ hybridization using a 30-basepair antisense oligonucleotide probe labeled with digoxigenin. Consistent with the above observation, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP treatment significantly augmented the total amount of irANP present in the cultures, with an ED50 of forskolin approximating 5 x 10(-5) M. Although treatment with 10(-7) M phorbol 12-myristate 13-acetate approximately doubled the production of irANP in the cultures (P less than 0.05), phorbol 12-myristate 13-acetate had little effect on modulating the number or neurite outgrowth of irANP neurons. Thus, our present findings suggest that protein kinase-A pathways are of greater importance than protein kinase-C pathways in regulating both the functional and morphological development of ANP-producing neurons during the ontogenesis of the rat hypothalamus.

XH1 – a new cervical carcinoma cell line and xenograft model of tumour invasion, 'metastasis' and regression

A new cell line, XH1, has been derived from an invasive focally keratinising adenosquamous carcinoma of the cervix in a 32 year old patient. It has been maintained in long term monolayer culture for 26 months, and passaged over 100 times (much greater than 300 population doublings). It is aneuploid with a mean chromosome number of 78. Examination using two minisatellite hypervariable DNA probes has shown it to be different from other cell lines maintained in this laboratory and from HeLa. Two sublines, XH1a and XH1b, show marked differences in monolayer culture, growth in soft agar, and xenograft formation. XH1 and XH1a cells readily form subcutaneous xenografts, and lung colonies can be established by their intravenous injection. Subcutaneous injection of XH1b cells results in rapid cell growth for a few days after which the tumour undergoes degeneration and then regresses completely. The XH1 karyotype has many rearranged chromosomes. Parental XH1 cells and both sublines show integration of HPV16 into the genome.

Forskolin-Induced Immunoreactive Atrial Natriuretic Peptide (ANP) Secretion and Pro-ANP Messenger Ribonucleic Acid Expression of Hypothalamic Neurons in Culture: Modulation by Glucocorticoids*

In the present studies the chronic effects of glucocorticoids and drugs activating cAMP-dependent pathways on the production and secretion of immunoreactive (ir) ANP from long term monolayer cultures of neonatal rat hypothalamic neurons were examined. Forskolin treatment increased ir-ANP release in a time-dependent and dose-related manner, with an EC50 of approximately 30 microM; at a lower dose of 10 microM, forskolin doubled ir-ANP release (P less than 0.01) compared to that in control cultures (mean +/- SEM, 9.6 +/- 0.3 pg/well; n = 4). While dexamethasone (DM) alone did not affect basal secretion of ir-ANP, 10 nM of the glucocorticoid significantly enhanced the effect of forskolin (10 microM) by raising ir-ANP release approximately 3 times that induced by forskolin alone (P less than 0.001). This potentiation of DM was both time dependent and dose responsive, with an EC50 of 1 nM; this effect was significantly suppressed by 100 nM RU38486, a glucocorticoid or type II receptor antagonist, but not by RU28318, a mineralocorticoid receptor antagonist. In addition, forskolin (10 microM) or DM (10 nM) alone significantly increased ir-ANP production approximately 1.4 times (P less than 0.05) and 1.3 times (P less than 0.05) over that of control cultures, respectively, whereas concurrent treatment with forskolin and DM increased ir-ANP production by approximately 1.8 times (P less than 0.01). These changes were reflected by a corresponding increment in the abundance of pro-ANP mRNA in the cultures, as demonstrated by Northern blot analysis. We conclude from the present findings that glucocorticoid- and cAMP-dependent pathways may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.

The Effects of Long Term Monolayer Culture on the Proteoglycan Phenotype of a Clonal Population of Mature Human Malignant Chondrocytes

Articular cartilage chondrocytes maintain biosynthetic heterogeneity in cell culture, but undergo irreversible dedifferentiation of their proteoglycan phenotype, as defined by keratan sulfate content. A recently described cell line of malignant human chondrocytes, 105KC, was the first to maintain a differentiated keratan sulfate-proteoglycan phenotype in long-term culture. A clone of 105KC, labeled KC2H3, is currently described and represents a distinct and metabolically more homogeneous population of mature chondrocytes than 105KC. KC2H3 cells universally express keratan sulfate biosynthesis, as defined by indirect immunofluorescence. In addition, KC2H3 expresses a more mature proteoglycan phenotype than 105KC, as demonstrated by the keratan sulfate content: 24% of glycosaminoglycan content of the aggregating proteoglycans of KC2H3 versus 13% for 105KC. Further reported are the effects of long term monolayer culture on the proteoglycan phenotype expressed by KC2H3. After more than 16 months in continuous monolayer, KC2H3 cells remained morphologically indistinguishable from those maintained in suspension alternating with monolayer. In addition, the proteoglycan phenotype remained mature, without a tendency towards dedifferentiation. The flattened morphology adopted by chondrocytes while in monolayer has been considered a stimulus of dedifferentiation; the present study is the first to examine the direct effects of physical state on a homogeneous and stable population of chondrocytes.

EVIDENCE FOR POST-TRANS LATI ON AL PROCESSING OF AURICULIN B TO ATRIOPEPTIN III IMMEDIATELY PRIOR TO SECRETION BY HYPOTHALAMIC NEURONS IN CULTURE

In rats, the precursor of atrial natriuretic peptide (ANP) is produced and processed into its smaller congeners in the heart and the brain. We have demonstrated that a congener of ANP, auriculin B (ANP4-28), was present in long term monolayer cultures of neonatal rat hypothalamic neurons. In addition, forskolin and 3-isobutyl-1-methyl-xanthine (IBMX) augmented the cellular content of auriculin B in a dose dependent manner. Thus, cyclic AMP may act as an intracellular signal for modulating the functional development of hypothalamic immunoreactive (ir) ANP producing neurons. Furthermore, our data suggest that the form of ANP released from these cultures, following either forskolin treatment alone or forskolin treatment followed by acute high potassium depolarisation, was atriopeptin III (ANP5-28). Thus atriopeptin III, rather than auriculin B, may represent the endogenous ligand for ANP receptors in the central nervous system.

The Production of Transforming Growth Factor-β by Chick Growth Plate Chondrocytes in Short Term Monolayer Culture*

Transforming growth factor-beta (TGF beta) is capable of regulating the proliferation and phenotypic expression of growth plate chondrocytes in culture. Chondrocytes were isolated from the growth plates from the long bones of 3- to 5-week-old chicks. Conditioned medium was harvested from short term monolayer cultures for the assay of TGF beta production by these cells. A receptor competition assay using [125I]TGF beta was used to quantitate the amount of TGF beta in the conditioned medium. Acid-activated conditioned medium contained 5.0 +/- 0.4 ng/ml TGF beta, while conditioned medium that had not been exposed to acid had undetectable levels of the peptide by this assay. The initial cell plating density was inversely related to the amount of TGF beta produced on a per cell basis. Growth plate chondrocytes separated by countercurrent centrifugal elutriation into maturationally distinct subpopulations had different rates of TGF beta production; hypertropic chondrocytes produced significantly more TGF beta (4.5 ng/10(6) cells) than the smallest chondrocytes isolated (2.3 ng/10(6) cells). A variety of other growth mediators were tested for their ability to influence TGF beta production by chondrocytes, and it was found that only basic fibroblast growth factor could significantly influence TGF beta production, producing a 6-fold increase in TGF beta recovered in the conditioned medium. The production of TGF beta by growth plate chondrocytes implicates it as an important autocrine or paracrine regulator in the process of endochondral calcification.

Proliferating Human Granulosa-Lutein Cells in Long Term Monolayer Culture: Expression of Aromatase, Cholesterol Side-Chain Cleavage, and 3β-Hydroxysteroid Dehydrogenase*

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of cholesterol side-chain cleavage and 17 alpha-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture.

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

Physiological concentrations of L-leucine control the release of prolactin from cultured rat pituitary cells.

In an attempt to further define the optimum experimental conditions for the in vitro study of PRL endocrinology, the effect of extracellular leucine concentration on PRL secretion was assessed. Rat pituitary cells, maintained in short term monolayer culture, were washed free of serum and placed in Minimum Essential Medium (MEM; 0.39 mM leucine) supplemented with nonessential amino acids and 0.1% gelatin. After the establishment of a constant rate of basal PRL release, the MEM was replaced with leucine-free MEM (supplemented as above), and the rate of PRL release was followed. PRL release from the cells in the leucine-free medium fell within 15 min to a rate 30% of that seen in the controls. Return to the control rate was achieved by the addition of L-leucine only. The D-isomer, isoleucine, valine, proline, leucyl-leucine, and leucine-agarose were ineffective. In the short term, the rate of PRL synthesis was found to be independent of extracellular leucine and the inhibition of release seen upon removal of leucine, and the subsequent recovery upon replacement of leucine was also observed when this experiment was conducted in the presence of 50 micrograms/ml cycloheximide. The L-leucine recovery was found to be dose dependent, with an increase in leucine concentration from 20 to 40 mg/liter causing a tripling of the PRL release rate. The addition of 10(-7) M TRH showed that leucine can be limiting in the response to this stimulant and allowed a response to as little as 10 mg/liter leucine to be discerned. From these experiments, it is concluded that physiological concentrations of leucine specifically and profoundly affect the release of PRL by a mechanism independent of protein synthesis. This leucine requirement for release has not been previously reported.

Insulin receptors of human term placental cells and choriocarcinoma (JEG-3) cells: characteristics and regulation.

Insulin has been implicated as a regulator of placental growth and function. In order to probe hormoneplacental interactions, an in vitro human placental cell culture model was employed in conjunction with the human choriocarcinoma cell line, JEG-3. Short term monolayer cultures of human term placentae were established from trypsinized chorionic villi and were presumed to be predominantly of syncytial origin on the basis of their electron microscopic morphology, their capacity to produce placental hormones, and their loss of replicative potential. Specific binding of [125I]iodoinsulin to both cell types was examined using whole cell radioreceptor assays, and was found to be a saturable process, time and temperature dependent, and proportional to cell protein and ligand concentration. At 4 C, a steady state was reached by 18 h and was maintained for 6 h. Specificity of the insulin receptor was evidenced by the inability of structurally unrelated hormones to inhibit [125I]iodoinsulin binding. Scatchard plot...

Muscarinic cholinergic stimulation of somatostatin secretion from long term dispersed cell cultures of fetal rat hypothalamus: inhibition by gamma-aminobutyric acid and serotonin.

Regulation of somatostatin (SS) secretion was studied in an in vitro system using collagenase-dispersed cells from fetal rat hypothalamus maintained in long term monolayer culture. Cultured cells exhibit a measurable basal secretion of immunoactive SS (SSLI) which can be augmented by carbachol, acetylcholine, or oxotremorine. The EC50 for carbachol is about 1 microM. Atropine, but not hexamethonium, antagonizes the action of cholinergic agonists. Cobalt or tetrodotoxin pretreatment diminishes basal secretion and eliminates the response to carbachol. Serotonin, several serotonin agonists, and gamma-aminobutyric acid (GABA) suppress carbachol-induced secretion. The GABA blockers bicuculline or picrotoxinin reverse the effect of added GABA and by themselves also augment SSLI secretion. Picrotin is inactive. The direct response to either bicuiculline or picrotoxinin is prevented by cobalt or tetrodotoxin treatment. These observations are consistent with the presence of a muscarinic cholinergic receptor which acts by a mechanism depending on an action potential and calcium influx to enhance the release of SSLI from neurosecretory cells. The data also support the conclusion that GABAergic transmission occurs within the cultures to tonically inhibit SSLI secretion. GABAergic, cholinergic, and serotoninergic systems may thus interact at the level of the hypothalamus to modulate SS secretion in vivo and thereby influence anterior pituitary release of GH and TSH.

Interaction of prolactin, estrogen and progesterone in a human mammary carcinoma cell line, cama-1—I. cell growth and thymidine uptake

The growth and thymidine uptake of a mammary carcinoma cell line (CAMA-1) in long term monolayer culture were investigated. These cells were maintained in the log phase for 7 days in Eagles' MEM medium with Earles' salts, supplemented with 15 or 25% fetal bovine serum (FBS) without further feeding. The growth rate of cells increased proportionally according to the amount of serum added (0–25%) and further increase was facilitated by 1 nM 17β estradiol added either to FBS or dextrancharcoal treated FBS (DCFBS). CAMA-1 cells propagated better in a 10% serum substitute (SS) plus 1% DCFBS than in 10% DCFBS. However, estrogen induced a smaller increase in cell growth in the former condition than in the latter. Prolactin (ovine) had no stimulatory effect on cell growth by itself or in combination with estradiol. Estrogen stimulated a dose-dependent increase of thymidine (TDK) uptake into cells: the stimulation was not observed in straight SS unless 1% or more FBS or DCFBS was added. In the absence of serum, prolactin or insulin had a permissive action for estrogen-induced TDK uptake. Progesterone added with or without estradiol had a slight proliferative activity on CAMA-1 cells at low levels but was inhibitory at high levels (10 μM) when cultured in SS condition. These results show that (a) CAMA-1 cells are estrogen-dependent, and respond to prolactin and progesterone in TDR uptake in culture, (b) estrogenic effect on cell growth and TDK uptake may require serum factors, (c) synergism or antagonism of estrogen action by prolactin and progesterone occurs in human breast cancer cells in culture, and (d) CAMA-1 cell line is a good alternative model for the investigation of hormonal interaction.

Regulation of Thyrotropin (TSH) Release and Production in Monolayer Cultures of Transplantable TSH-Producing Mouse Tumors1

Studies of TSH release and production were performed in short term monolayer cultures of transplantable, thyroid hormone responsive, thyrotropin (TSH) producing mouse pituitary tumors. These tumors contained large amounts of TSH, small amounts of growth hormone (GH) and no detectable luteinizing hormone (LH), indicating that the predominant hormone product of tumor cells was TSH. The TSH content per tumor cell was similar to that of the normal pituitary where thyrotrophs represent a small fraction of the total cells, suggesting that the TSH content per tumor cell was less than that of the normal thyrotroph. There was a time dependent release and production of TSH by tumor cells in monolayer culture. Thyrotropin releasing hormone (TRH) increased the release into the media and the production of TSH in a dose dependent manner. Maximum effects were noted at 0.2 ng/ml. Thyroid hormones and somatostatin inhibited both basal and TRH induced effects on both TSH release and production. TSH release as induced by TRH was calcium dependent. TSH release was stimulated by ouabain (10(-3)M) and potassium (57 mM), agents known to promote cellular calcium uptake in a calcium dependent manner. These studies indicate that tumor derived cells function in monolayer culture in a similar fashion to normal thyrotrophs. Studies were conducted to test the hypothesis that TRH action is mediated by adenosine 3',5' monophosphate (cAMP). Dibutyryl cAMP (6 mM) and theophylline (10 mM) increased TSH release suggesting that cAMP is involved in TSH release. However, TRH had no detectable effect on tumor cell adenylate cyclase activity or levels of cAMP. In contrast, PGE1 (1-10 mug/ml) stimulated adenylate cyclase activity and elevated cellular levels of cAMP without increasing TSH release. Thus, we are unable to confirm the postulate that cAMP is the intracellular mediator of TRH action.