Thymidine Assay Research Articles

Studies on the differentiation pathway and growth characteristics of epithelial culture cells of the human prostate.

We established explant primary cultures in order to study the growth and hormone responsiveness, and the differentiation process of prostatic epithelial cells. Cell outgrowth was achieved from explant tissue by using a new DU145-cell-conditioned medium and special plastic coverslips. To define the present model, proliferation assays were tested by [3H]thymidine assay and planimetric analysis. Cells were analyzed using immunocytochemistry, light, phase contrast and electron microscopy, polymerase chain reaction, telomerase ELISA and immunoassay (PSA). Morphology and electron microscopy revealed typical epithelial differentiation. Immunocytochemistry showed the content of basal and secretory epithelial cells, endocrine paracrine cells and a high level of proliferation. With increasing culture time, mature epithelial differentiation (PSA) increases and the initial increase of alpha-smooth muscle actin (alpha-SMA) decreases again. After further passaging, alpha-SMA expression is no longer detected and PSA expression decreases. Furthermore, epithelial cells showed both androgen responsiveness and androgen receptor expression. These findings show the presence of epithelial cells in a process of differentiation with endocrine paracrine cells and a high level of proliferation. This model may maintain the cellular and functional properties more closely related to the human prostate and may provide a valuable tool for studying stem cells and differentiation characteristics.

Effect of keratinocyte growth factor and activin on cell growth in the human prostatic cancer cell line LNCaP.

Keratinocyte growth factor (KGF) has paracrine properties in the human prostate which stimulate epithelial cell growth. Activins have profound effects on cell growth and function in the human prostate, and are expressed in LNCaP, DU 145 and PC3 cells. LNCaP cells were characterized by immuncytochemistry, an immunoassay and polymerase chain reaction. A 3[H]thymidine assay was used with 0.01-10 nM dihydrotestosterone, 10 micro M flutamide, 1-100 ng/ml KGF and 3 nM activin. LNCaP cells expressed Ki67, PSA, cytokeratins (8, 18, 19, 14, 15) androgenreceptor but no KGF protein. LNCaP cells showed telomerase activity. Furthermore, ARmRNA (365 bp), but no KGF or KGFRmRNA were expressed. KGF ELISA detected no intracellular or secreted KGF. DHT (1, 10 and 100 nM) and KGF (10 and 100 ng/ml) significantly stimulated LNCaP cell proliferation. However, flutamide and 3 nM activin A significantly decreased cell proliferation in the presence and absence of KGF. The results of our experiments support the hypothesis that cell growth and proliferative characteristics of LNCaP cells are modulated by KGF and activin A.

Hepatocyte growth factor regulates proteoglycan synthesis in interstitial fibroblasts

Hepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects. Expression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P < 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-beta (TGF-beta)-induced changes in cell proliferation. These results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38 MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-beta inhibitory actions in the kidney.

996 Effect of retinoic acid analogue on tumor growth and angiogenesis

Gemcitabine is a first-line agent for advanced pancreatic cancer therapy. However, its efficacy is often limited by its poor intracellular metabolism and chemoresistance. To exert its antitumor activity, gemcitabine requires to be converted to its active triphosphate form. Thus, our aim was to improve gemcitabine activation using gene-directed enzyme prodrug therapy based on gemcitabine association with the deoxycytidine kinase::uridine monophosphate kinase fusion gene (dCK::UMK) and small interference RNA directed against ribonucleotide reductase (RRM2) and thymidylate synthase (TS). In vitro, cytotoxicity was assessed by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide and [3H]thymidine assays. Apoptosis-related gene expression and activity were analyzed by reverse transcription-polymerase chain reaction, Western blot, and ELISA. For in vivo studies, the treatment efficacy was evaluated on subcutaneous and orthotopic pancreatic tumor models. Our data indicated that cell exposure to gemcitabine induced a down-regulation of dCK expression and up-regulation of TS and RR expression in Panc1-resistant cells when compared with BxPc3- and HA-hpc2-sensitive cells. The combination of TS/RRM2 small interference RNA with Ad-dCK::UMK induced a 40-fold decrease of gemcitabine IC50 in Panc1 cells. This strong sensitization was associated to apoptosis induction with a remarkable increase in TRAIL expression and a diminution of gemcitabine-induced nuclear factor-κB activity. In vivo, the gemcitabine-based tritherapy strongly reduced tumor volumes and significantly prolonged mice survival. Moreover, we observed an obvious increase of apoptosis and decrease of cell proliferation in tumors receiving the tritherapy regimens. Together, these findings suggest that simultaneous TS/RRM2-gene silencing and dCK::UMK gene overexpression markedly improved gemcitabine's therapeutic activity. Clearly, this combined strategy warrants further investigation.

Role of α8 integrin in mesangial cell adhesion, migration, and proliferation

Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, migration, and proliferation. The alpha8 integrin chain is expressed in the glomerulus exclusively by mesangial cells. The contribution of alpha8 to mesangial cell function, however, has not yet been studied. Mesangial cells from wild-type and alpha8-deficient mice were isolated and characterized. Integrin expression was assessed by real-time polymerase chain reaction (PCR), Western blot, or fluorescence-activated cell sorter (FACS) analysis. Cell adhesion was determined by conventional attachment assay and a centrifugal assay for cell adhesion. Cell migration was determined by a fluorescence-based transmigration assay and a chemotaxis assay. Proliferation rates were determined by BrdU and [3H]-thymidine assays. On the alpha8 ligands fibronectin and vitronectin, but not on collagens, attachment of alpha8-deficient mesangial cells was reduced compared to wild-type cells. In contrast, alpha8-deficient mesangial cells migrated more easily and displayed an increased proliferative response on fibronectin or vitronectin, but not on collagens, compared to wild-type cells. These effects were not due to an up-regulation of the fibronectin or vitronectin receptors alpha5 or alphav in alpha8-deficient mesangial cells, as the cell surface expression of integrins alpha5 and alphav was comparable in wild-type and alpha8-deficient mesangial cells. These findings confirm a role for alpha8 integrin in the regulation of the mesangial cell phenotype. alpha8 integrin seems to promote adhesion, but inhibit migration and proliferation of mesangial cells. Thus, the data support the hypothesis that alpha8 integrin could play an important role for maintaining tissue integrity in the glomerulus during glomerular injury.

Variability in zinc tolerance, measured as incorporation of radio-labeled carbon dioxide and thymidine, in periphyton communities sampled from 15 European river stretches.

Fifteen European rivers and streams belonging to watersheds in Sweden, the Netherlands, and Spain respectively, were sampled by allowing periphyton to colonize submerged glass substrata. Their zinc tolerances were quantified in short-term laboratory tests, where inhibition of photosynthesis in microalgae and thymidine incorporation in bacterial DNA was measured, and expressed as EC50 values. The variability in zinc tolerances was high reaching 1.5-2.5 orders of magnitude, ranging from 25-8145 microM for photosynthesis and 15-467 microM for thymidine assays. Based on the observed variability, uncertainty factors were estimated for the extrapolation of zinc toxicity data from river to river, both regionally and interregionally. Under the assumption to protect 95% of the observed communities the regional uncertainty factors were 1.7-4.3 and the interregional 2.4-8.6. The sampling sites were characterized in terms of biotope physiography, water chemistry, periphyton biomass, trace element content, and species composition. Multivariate analysis of the data using PLS (Projection to Latent Structure), was used to generate hypotheses about the relation between periphyton zinc tolerance and the 123 so-called predictor variables. Zinc contamination, phosphate, nitrogen nutrients, pH, calcium, bicarbonate, dissolved organic carbon, and various diatom species are important predictors for zinc tolerance in the entire data set representing all 15 river stretches. Regional models suggested that very different factors determined the zinc tolerance in the Swedish and Dutch periphyton. The results are interpreted in terms of Pollution-Induced Community Tolerance (PICT) and the bioavailability of zinc.

Differential effects of insulin-like growth factor binding proteins-1, -2, -3, and -6 on cultured growth plate chondrocytes

In children with chronic renal failure (CRF), impairment of longitudinal growth is in part due to excess amounts of circulating high-affinity insulin-like growth factor binding proteins (IGFBPs) that might decrease or prevent insulin-like growth factor (IGF) binding to its signaling receptor. However, it appears from the clinical studies that various IGFBPs may have contrasting effects on longitudinal growth. Because of the potential importance of the IGFBPs as modulators of longitudinal growth in pediatric CRF, the aim of the present study was to investigate the biological effects of IGFBP-1, -2, -3, and -6 on cultured growth plate chondrocytes that express the type 1 IGF receptor. The effects of exogenous IGFBPs on IGF-independent and IGF-dependent proliferation of rat growth plate chondrocytes in primary culture were investigated. Proliferation was assessed by colony formation of agarose-stabilized long-term suspension cultures and by the [3H]thymidine assay. The effects of IGFBPs on IGF-I binding and the binding of IGFBPs to chondrocytes were assessed by binding studies with radiolabeled proteins in monolayer culture. Intact IGFBP-1, IGFBP-2 and IGFBP-6 inhibited in equimolar concentration the IGF-I- and IGF-II-stimulated DNA synthesis and cell proliferation, whereas the biological activity of IGFBP-3 was complex. It had an IGF-independent antiproliferative effect and also inhibited IGF-dependent chondrocyte proliferation under coincubation conditions, whereas under preincubation conditions IGFBP-3 enhanced IGF-I-responsiveness. Studies on the mechanism by which IGFBP-3 potentiated IGF activity demonstrated that under preincubation conditions IGFBP-3 is capable to associate with the cell membrane and to facilitate IGF-I cell surface binding. Intact IGFBP-1, IGFBP-2 and IGFBP-6 act exclusively as growth inhibitors on IGF-dependent proliferation of growth plate chondrocytes. IGFBP-3, however, can either inhibit IGF-independent and IGF-dependent cell proliferation, or enhance IGF responsiveness of chondrocytes dependent on the temporal relationship to the IGF exposure.

Human B Lymphocytes and B Lymphomas Express PPAR-γ and Are Killed by PPAR-γ Agonists

This paper evaluates the expression and functional significance of PPAR-γ on human B cells. Recent interest in PPAR-γ has focused on its adipogenic effects on non-bone marrow-derived cells. PPAR-γ agonists also have been proposed as anti-inflammatory agents owing to inhibition of NF-κB activation. We report herein the first study evaluating PPAR-γ expression and functional significance in human B lineage cells. Interestingly, normal human B cells and a variety of B lymphoma cells (e.g., Daudi, Ramos, and Raji) express PPAR-γ protein as determined by immunocytochemistry. The expression of 80-kDa PPAR-γ on human B lymphocytes and B lymphomas was confirmed by Western blot analysis. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), a natural PPAR-γ agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [3H]thymidine and MTT assays. Only PPAR-γ agonists (thiazolidinediones) and not PPAR-α agonists mimicked the effect of 15d-PGJ2 on B lineage cells, indicating that the mechanism by which 15d-PGJ2 negatively affects B lineage cells involves, in part, PPAR-γ. The mechanism whereby PPAR-γ agonists induce cytotoxicity is via apoptosis as shown by Annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. This is the first study evaluating PPAR-γ expression and its significance on human B lymphocytes. PPAR-γ agonists may serve as a counterbalance to the stimulating effects of other prostaglandins, namely PGE2, which promotes B cell immunoglobulin class switching. Finally, the use of prostaglandins such as 15d-PGJ2 and synthetic PPAR-γ agonists to induce apoptosis in B lineage cells may lead to the development of novel therapies for potentially fatal B lymphomas.

The Influence of Fibronectin and/or RGDS Tetrapeptide on Osteopontin Expression in Cultures of Rat Calvarial Osteoblasts

Purpose : To investigate the effect of fibronectin (FN) and/or RGDS tetrapeptide on osteoblastic proliferation and osteopontin expression. Materials and Methods : Osteoblasts were cultured on tissue culture polystyrene (TCPS) and apatite-wallastonite glass ceramics (AW-GC) with or without FN and RGDS synthetic tetrapeptide. Osteoblastic proliferation and differentiation were measured after cultivation, by determining the number of attached cells, by []-thymidine assay, and by measuring the activity of alkaline phosphatase. The expression of osteopontin (OPN) was determined by RT-PCR and western blotting. Results : Cellular proliferation was more increased by FN than the other variables examined (P

Induction of Myocarditis and Valvulitis in Lewis Rats by Different Epitopes of Cardiac Myosin and Its Implications in Rheumatic Carditis

Immune responses against cardiac myosin and group A streptococcal M protein have been implicated in the pathogenesis of rheumatic heart disease. Although cardiac myosin is known to produce myocarditis in susceptible animals, it has never been investigated for its role in production of valvular heart disease, the most serious sequelae of group A streptococcal infection in acute rheumatic fever. In our study, cardiac myosin induced valvulitis in the Lewis rat, and epitopes responsible for production of valvulitis were located in the rod region. Human and rat cardiac myosins induced severe myocarditis in the Lewis rats as expected. A purified S2 fragment (amino acid sequences 842 to 1295) produced the most severe myocarditis as well as valvulitis. Different regions of light meromyosin produced valvulitis (residues 1685 to 1936) or myocarditis (residues 1529 to 1611). Because streptococcal M proteins produced valvular heart disease in Lewis rats and have been linked to anti-cardiac myosin responses, we reacted myosin-sensitized lymphocytes isolated from the hearts of Lewis rats with peptides of streptococcal M5 protein in tritiated thymidine assays. Infiltrating lymphocytes responded most strongly to peptides within the B repeat region of streptococcal M protein. These data show direct evidence that immune responses against cardiac myosin lead to valvular heart disease and the infiltration of the heart by streptococcal M protein reactive T lymphocytes.

Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT 2 transporter protein and mRNA of INS-1 cells.

Both 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA) are active metabolites of vitamin A (retinol). There exists an interaction between retinoid receptors and peroxisome proliferator-activated receptors (PPARgamma). To define their functions in an insulin secreting system the effects of ATRA, 9cRA and the PPARgamma agonist rosiglitazone on cell proliferation, insulin release and glucose transporter (GLUT) 2 of INS-1 cells were tested. Retinoic acid receptor (RAR-alpha and -gamma) and retinoid X receptor (RXR-alpha and -beta) proteins are present (immunoblots). Both 9cRA and ATRA inhibit INS-1 cell proliferation ([3H]-thymidine assay) in a concentration dependent manner. Both 9cRA and ATRA increased insulin release, but only ATRA ralsed the GLUT 2 mRNA in a bell-shaped concentration response curve after 48 h. The insulinotropic effect of one compound is not significantly superimposed by the other indicating that the same binding sites are used by 9cRA and ATRA. The acute and chronic effects of the PPARgamma agonist rosiglitazone on insulin release were additionally determined since glitazones act as transcription factors together with RXR agonists. At high concentrations (100 microM) rosiglitazone inhibited glucose (8.3 mM) stimulated insulin secretion (acute experiment over 60 min). Insulin secretion, however, was increased during a 24 h treatment at a concentration of 10 microM and again inhibited at 100 microM. Changes in preproinsulin mRNA expression were not observed. Rosiglitazone (100 microM) increased GLUT 2 mRNA paralleled by an increase of GLUT 2 protein, but only after 24 h of treatment. This data indicate that RAR and RXR mediate insulin release. The changes in GLUT 2 have no direct impact on insulin release; the inhibition seen at high concentrations of either compound is possibly the result of the observed inhibition of cell proliferation. Effects of rosiglitazone on preproinsulin mRNA and GLUT 2 (mRNA and protein) do not play a role in modulating insulin secretion. With the presence of an RXR receptor agonist the effect of rosiglitazone on insulin release becomes stimulatory. Thus the effects of RAR-, RXR agonists and rosiglitazone depend on their concentrations, the duration of their presence and are due to specific interactions.

Comparative effects of indomethacin on cell proliferation and cell cycle progression in tumor cells grown in vitro and in vivo

Considerable research effort is currently being directed towards understanding the mechanisms mediating the antiproliferative effects of non-steroidal anti-inflammatory drugs (NSAIDs) and, more recently, of cyclooxygenase (COX)-2 inhibitors as well. A key question is whether NSAIDs (excluding sulindac) exert their anticarcinogenic effects in vivo by a mechanism that is dependent on their capacity to inhibit COX activity. Some studies with cultured tumor cells in vitro have argued against such a linkage, showing that NSAIDs inhibit cell replication and/or augment apoptosis only at concentrations that exceed those required to inhibit COX activities 10- to 100-fold. The significance of these results for the observed anticarcinogenic effects of NSAIDs in vivo has not yet been evaluated. We addressed this question by comparing, for the same tumor cells, the effects of the NSAID indomethacin on cell growth parameters when the cells were grown in culture to the effects seen in the in vivo growing tumor in the mouse. Indomethacin added to cultured Lewis lung carcinoma cells exerted a potent antiproliferative effect ( 3H thymidine assay) and reduced cell viability (MTT[3-(4,5-dimethyl(thiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay) at low doses (10–20 μM) in parallel with its inhibitory effect on cellular cyclooxygenase. These effects of indomethacin appeared to arise from a clear antiproliferative shift in the profile of the cell cycle parameters towards a reduced percentage of cells at the S and G 2/M phases, together with an increased percentage of cells at the G 1 phase. Significantly, similar results were seen when indomethacin was given in vivo at the low dose of 2 mg per kg/day, which blocked blood platelet COX activity and at the same time produced a delay in tumor growth initiation and attenuation of apparent primary tumor growth as well as growth of lung metastases. These results thus provide strong support for the notion that COX inhibition is a major determinant in the antitumorigenic effect of indomethacin in vivo.

Tyrolobibenzyls - Novel Secondary Metabolites fromScorzonera humilis

Tyrolobibenzyls A (2), B (3), and C (4) were isolated from Scorzonera humilis (Asteraceae) of Tyrolean origin. Compounds 2 and 3 possess a unique phenylethyl-benzofuran backbone and represent a new class of natural compounds. Structural assignments are based on 1D and 2D NMR as well as MS data. Furthermore, peracetylated derivatives of compounds 3 and 4 were synthesized. Pharmacological evaluation of these compounds by the [3H]thymidine assay showed no pronounced cytotoxicity.

Melatonin decreases cell proliferation and transformation in a melatonin receptor-dependent manner

There are conflicting claims for the role of melatonin in oncogenesis. In addition, the mechanism(s) underlying melatonin's effects in oncogenic processes is (are) unknown. In this study, the effects of melatonin exposure on cell proliferation and transformation were assessed in NIH3T3 cells transfected with either the human mt 1 (NIH-mt1) or MT 2 (NIH-MT2) melatonin receptors. The effects of melatonin exposure on proliferation was assessed by direct cell counts and [ 3H]thymidine uptake assays. The effect of chronic melatonin pretreatment on transformation was assessed by focus assays. In both NIH-mt1 and NIH-MT2 cells, melatonin pretreatment decreased cell proliferation and transformation. Control (NIH-neo) cells did not show this effect. However, as revealed by the [ 3H]thymidine uptake assays, an increase in DNA synthesis occurred in NIH-mt1 cells, whereas no increase occurred in the NIH-MT2 or NIH-neo cells. Upon examination of melatonin receptors, a decrease in the function of both mt 1 and MT 2 receptors occurred. These data suggest that perhaps an attenuation of receptor-mediated processes are involved in the anti-proliferative and anti-transformation capabilities of melatonin in NIH3T3 cells. In addition, based on the [ 3H]thymidine assays, receptor mediated signal transduction mechanisms may slow the growth of cells via actions on the cell cycle. The results from this study shed new insight on the putative mechanisms underlying melatonin's effects on cell proliferation and transformation and lends support for a protective role of melatonin in oncogenesis.

CHARACTERIZATION OF A STROMAL CELL MODEL OF THE HUMAN BENIGN AND MALIGNANT PROSTATE FROM EXPLANT CULTURE

Purpose There is a lack of suitable in vitro models for the human prostate. To study stromal-epithelial interactions, we established stromal cells in cultures from benign and malignant prostate tissue that resemble more closely the in vivo conditions of the human prostate. Materials and Methods Stromal cells were obtained from explant primary culture, established in DU145 cell conditioned medium and maintained in RPMI-fetal bovine serum (FBS) supplemented with insulin, transferrin and selenium (ITS). Proliferation studies to compare different media were performed using a 3 [H]thymidine assay. Stromal cells were characterized by immunocytochemistry using epithelial and mesenchymal markers. Morphology was evaluated by electron microscopy, light and phase-contrast microscopy. Androgen receptor (AR) mRNA expression was measured by polymerase-chain-reaction (PCR). The response to different concentrations of dihydrotestosterone (DHT) and the antihormones flutamide and hydroxyflutamide was tested by 3 [H] thymidine assay. Results Microscopic evaluation revealed typical stromal morphology with elongated cell shapes, cilia, collagen and microfilaments. Immunocytochemical characterization revealed typical fibroblastic and smooth muscle differentiation. ITS supplemented in RPMI-FBS showed the best growth stimulation compared with other serum-free media (p <0.05) and became our basal medium. The presence of DU145 cell conditioned medium in this basal medium showed a significant increase in cell proliferation in stromal cells. Stromal cells maintained AR mRNA expression and significant DHT dose dependent growth stimulation in up to 10 passages. Both the antiandrogens flutamide and hydroxyflutamide counteracted the DHT effect (p <0.05). Conclusions This stromal cell model maintains many cellular and functional properties of the human prostate, which may enable us to study growth factor modulation, drug and hormone metabolism in stromal-epithelial interaction with emphasis on the pathogenesis of BPH and prostate cancer.

Detection of Ob-receptor in human adrenal neoplasms and effect of leptin on adrenal cell proliferation.

Leptin, a hormone mainly secreted from adipose tissue, communicates a metabolic signal to the adrenal gland. Ob-Receptor (Ob-R) expression was reported in rat, mice and human adrenal glands. This study intended to investigate possible differences in the Ob-R expression and distribution of Ob-R protein in human adrenal tumors as compared to normal adrenal tissue. Proliferative effects of leptin were analyzed in the human adrenocortical carcinoma cell line (NCI-H295). The full length Ob-R mRNA and the isoforms B219.1 and B219.3 could be demonstrated by RT-PCR in all adrenal tumors (n=8), the tumor cell line (NCI-H295) and normal tissue. In contrast the Ob-R isoform B219.2 was absent in the carcinoma cell line and in most of the adrenal tumors (n=5), whereas it was present in normal adrenals. The Ob-R protein could be demonstrated in benign and malignant adrenocortical tumors. Pheochromocytomas showed only a weak immunostaining with the human Ob-R antibody. Human leptin did not affect the proliferation or variability of adrenal tumor cells as demonstrated by [3H]-thymidine assay and WST-1 test. In conclusion, although functional leptin receptors are expressed in human adrenal tumors, leptin does not regulate tumor cell proliferation.

Differential Inhibitory Effects on Human Endometrial Carcinoma Cell Growth of Luteinizing Hormone-Releasing Hormone Analogues

In addition to its function as a key hormone in the regulation of the pituitary–gonadal axis, luteinizing hormone-releasing hormone (LHRH) probably also affects various extrapituitary tissues. LHRH binding sites andin vitroantiproliferative effects of LHRH analogues have been reported in human endometrial cancer. The effects of the LHRH agonist leuproreline and LHRH antagonist antide were studied on the cell growth, DNA synthesis, and cell cycle distribution of the human endometrial cancer cell lines HEC-1A and HEC-1B by the sulforhodamine B (SRB) method, [3H]thymidine assay incorporation, and propidium iodide DNA staining, respectively. In the presence of 1.0–100 μM leuproreline the proliferation of HEC-1A cells was significantly reduced as early as 3 days after drug exposure, with a minimum growth value of 69.9 ± 3.6% (mean ± SE) at the highest concentration tested (100 μM). Similar antiproliferative effects were obtained following a 6-day treatment with the LHRH antagonist antide. Also, inhibitory effects on [3H]thymidine incorporation into the DNA of the HEC-1A cell line were noted after a 6-day exposure to both LHRH analogues, in the above-mentioned concentration range. Cell cycle analysis of HEC-1A cells cultured in the presence of 10 μM leuproreline and antide showed a slight accumulation of cells in the G0/G1phase, while the proportions of cells in the S and G2/M phases concomitantly decreased. No significant effects on proliferation, DNA synthesis, and cell cycle distribution were observed in HEC-1B cells with either leuproreline or antide (up to 100 and 10 μM, respectively) after a 6-day exposure. Both Northern blot analysis and reverse transcription polymerase chain reaction failed to detect expression of mRNA for the LHRH receptor in both HEC-1A and HEC-1B cell lines. In addition, the LHRH analogues did not affect the intracellular free calcium concentration, indicating that the classic signal transduction for LHRH is absent or impaired in HEC-1A cells. The observed direct inhibitory actions on HEC-1A cells support the concept that the two LHRH analogues may exert biological effects via cellular effectors distinct from the “classic” LHRH receptor. Although the mechanism by which these direct actions are produced is still obscure, these results might help to establish the basis for new approaches to the therapy of endometrial cancer.

Bacterial growth dynamics in activated sludge batch assays

Aerobic batch reactors are often used to estimate the maximum specific growth rates of bacteria in wastewater treatment environments. Results from these reactors vary, and they are sometimes confusing because they do not represent the bacterial populations in the original wastewater environment, i.e. in situ. For the first time in batch reactors, we present the in situ growth dynamics of these bacterial populations. We simultaneously measured bacterial numbers and growth rate, oxygen uptake rates (OUR), soluble COD and biomass COD (mg.l −1). With a substrate to biomass (S o/X o) ratio of 1.2, bacterial specific growth rates, measured using the thymidine growth assay, increased from 1 to 4.5 d −1. During the same period, the initial OUR remained relatively constant, with an OUR-based estimate of the maximum specific growth rate of 3.6 d −1. These observations challenge the fundamental theory of the batch assay that bacteria are growing at their maximum rate under substrate saturating conditions while the yield coefficient remains constant. Our results confirm that the batch reactor environment induces changes in the bacterial physiology and/or community structure compared to the original treatment environment, even at low S o/X o ratios. We discuss how, simultaneously using both OUR and thymidine assays, gives more information about how the cell allocates its energy resources between new cell synthesis and cell maintenance.

Application and evaluation of the alamarBlue assay for cell growth and survival of fibroblasts.

Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. Various assay parameters were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20,000 cells/well, which is similar to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and [3H]thymidine assay techniques. Standard (reference) curves generated with a known fibroblast stimulator were used to facilitate quantitation and comparison of unknown test substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipate that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for investigating the complex mechanisms of cellular proliferation.

Lymphocytes of human term decidua decrease cell adhesion to a plastic substrate.

Although human decidual lymphocytes have been widely studied, their function and possible interaction with trophoblast are still unclear. Here we show that whereas human early (EDL) and term (TDL) decidual lymphocytes were unable to kill human trophoblast by necrosis (assessed by the 51Cr-release assay) or apoptosis (DNA fragment assay), TDL but not EDL decreased trophoblast adhesion to a plastic substrate as determined by a [3H]thymidine assay. This effect, however, was not selective for trophoblast, as TDL also decreased the adhesion to plastic of human decidual stromal cells and HeLa cells. Our results suggest that TDL may play a role in placental detachment during parturition by decreasing trophoblast or decidual stromal cell adhesion.