Incorporation Of Radiolabeled Thymidine Research Articles

T lymphocyte phenotype of contact-allergic patients: experience with nickel and p-phenylenediamine.

There is considerable interest in understanding the immunological variables that have the greatest influence on the effectiveness of sensitization by contact allergens, particularly in the context of developing new paradigms for risk assessment of novel compounds. To examine the relationship between patch test score for three different contact allergens and the characteristics of T cell responses. A total of 192 patients with confirmed nickel, p-phenylenediamine (PPD) or methylisothiazolinone (MI) allergy were recruited from the Contact Dermatitis Investigation Unit at Salford Royal Hospital. Severity of allergy was scored by the use of patch testing, peripheral blood lymphocytes were characterized for T cell phenotype by flow cytometry, and proliferative activity was characterized by radiolabelled thymidine incorporation. Comparisons were drawn with buffy coat samples from healthy volunteers. Patch test positivity for nickel, PPD and MI was associated with changes in the phenotype of peripheral blood T cells: increases in naïve cells, decreases in regulatory T cell frequency and the CD4+ /CD8hi ratio, and increased expression of the skin-homing marker cutaneous lymphocyte antigen (CLA), particularly for those patients with a +++ patch test score. This increased understanding of the characteristics of the T cell responses to contact allergens may provide parameters with which to better measure health risks associated with skin sensitization.

Activation of β2-adrenergic receptor by (R,R′)-4′-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells

(R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a highly-selective β2 adrenergic receptor (β2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R′)-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R′)-MNF positively correlated with the β2-AR expression levels across tested cell lines. The anti-motility activity of (R,R′)-MNF was inhibited by the β2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R′)-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R′)-MNF caused significant inhibition of cell growth in β2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon β2-AR activation with (R,R′)-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified β2-AR as a novel potential target for melanoma management, and (R,R′)-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in β2-AR-expressing lesions.

The interplay of human chorionic gonadotropin (hCG) with basic fibroblast growth factor and adipokines on angiogenesis in vitro

IntroductionHuman chorionic gonadotropin (hCG) is suggested to regulate placental angiogenesis, however, its role is incompletely understood. hCG may directly stimulate angiogenesis or influence the effect of other angiogenic factors. We examined the effect of hCG and the interplay of hCG with basic fibroblast growth factor (bFGF) and with various adipokines on proliferation of vascular endothelial cells in vitro. MethodsHuman umbilical vein endothelial cells (HUVEC) were incubated for 2 days with combinations of hCG, bFGF, leptin, resistin, adiponectin, IL6 and TNFα. Incorporation of radiolabelled thymidine was used to assess cell proliferation. Immunofluorescence and flow cytometry were used to examine activation of p44/42 mitogen-activated kinase (MAPK). ResultshCG induced proliferation of HUVEC alone and in combination with bFGF. Cells exposed to both hCG and bFGF displayed increased activation of p44/42 MAPK as compared to hCG or bFGF alone. Increased HUVEC proliferation was observed in the presence of increasing concentrations of leptin, resistin, adiponectin, and IL6, whereas HUVEC proliferation decreased in the presence of TNFα. hCG in combination with leptin, resistin, adiponectin or IL6 stimulated HUVEC proliferation beyond the effect of hCG alone. DiscussionAn interplay of hCG with adipose tissue-derived factors with angiogenic properties is plausible. Thus, maternal obesity may affect placental angiogenesis in pregnancy. ConclusionshCG may directly stimulate angiogenesis. Also, hCG may indirectly stimulate angiogenesis through interplay with bFGF and adipokines.

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

BackgroundIt is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.MethodsThe rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.ResultsIn the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.ConclusionThe results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).

Bacterial Physiological State in Wastewater: Monitoring Maintenance and Production with Leu/TdR Ratio for Less Pollution

In domestic wastewater, bacterial physiology controls cell production (growth, replication) and cell maintenance, determining how energy is allocated between these two processes. The aim here was to develop a method to quantify these cellular processes so that the bacterial physiological state could be manipulated to lower this source of pollution. We simultaneously used the incorporation of radiolabelled thymidine into DNA (a measure of new cell synthesis) and leucine into protein in wastewater to quantitatively distinguish bacterial growth from maintenance processes. We found that DNA and protein syntheses were coupled in wastewater after substrate enrichment (with glucose or acetate)—balanced growth. Once the substrate was depleted, the two processes became uncoupled—unbalanced growth. In this physiological state, the bacteria were synthesising protein, but fewer bacteria were replicating. More energy was allocated to cell maintenance than replication. A mean Leu/TdR ratio of 7.4 was determined for wastewater and was similar to natural aquatic ecosystems. As the bacterial growth rate decreased, the Leu/TdR ratios increased. We show how the simultaneous measurement of [3H]Leu and [3H]TdR quantitatively distinguishes balanced from unbalanced growth. Low [3H]Leu/[3H]TdR ratios indicated bacteria were physiologically stressed, an ideal state for biological wastewater treatment processes (WWTP) as the bacteria divert more energy to maintenance activities instead of growth. Leu/TdR ratios of 70 have been recorded in natural aquatic ecosystems which suggests WWTP have potential to be manipulated to achieve much higher Leu/TdR ratios than we report here. Changes to plant operation to improve operation efficiency include finding the optimum rate of substrate (pollution supply) or alternating aerobic and anaerobic periods to maximise the Leu/TdR ratio to achieve less biomass production for land disposal and more cost-effective operation that generates less pollution in the effluent.

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p<0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor-bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth in bone. Thus, targeting TGF-β receptor I is a valuable intervention in men with advanced PCa.

Abstract C146: In human lung carcinoma cells that contain estrogen receptor-α(ER), thyroid hormone-induced proliferation initiated at an integrin requires ER.

Abstract We examined the molecular basis of thyroid hormone-induced proliferation of two estrogen receptor-α (ER)-expressing human lung cancer cell lines, non-small cell carcinoma NCI-H522 and small cell carcinoma NCI-H510A. At a physiologic total L-thyroxine (T4) concentration (10[−7] M) and supraphysiologic 3,5,3'-triiodo-L-thyronine (T3) levels, thyroid hormone significantly increased proliferating cell nuclear antigen (PCNA) content of both cell lines. Neutralizing antibody to integrin αvβ3 and integrin-binding Arg-Gly-Asp (RGD) peptide inhibited cell proliferation stimulated by thyroid hormone. Tetraiodothyroacetic acid (tetrac) blocks binding of T4 and T3 to the hormone receptor on αvβ3 and inhibited thyroid hormone-induced cancer cell proliferation. Thus, the thyroid hormone effect is initiated nongenomically at the cell surface thyroid hormone receptor we have described on integrin αvβ3. The thyroid hormone isoforms also caused serine phosphorylation of ER in NCI-H522 and NCI-H510A cells. The ER antagonist, ICI 182,780 (fulvestrant) inhibited stimulation by T4 and T3 of ER phosphorylation, of PCNA accumulation and of radiolabeled thymidine incorporation by the cells. These results are consistent with existence of crosstalk between the plasma membrane receptor for iodothyronines and ER in these lung cancer cells. We conclude that endogenous T4 is a growth factor for ER-containing lung cancer cells and this hormonal action is subject to inhibition by tetrac and by fulvestrant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C146.

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

BackgroundNeurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.MethodsColon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.ResultsNeurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.ConclusionsWhile acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.

Effect of Chronic High Altitude Hypoxia on Foetal and Maternal Juxta-Alveolar Distal Pulmonary Smooth Muscle Cells Actin and Calponin Organisation and Growth Profiles

The effect of chronic high altitude hypoxia (CHAH) in the juxta-alveolar region near the air-blood interface is unknown because of the experimental inaccessibility of this region. To examine primary cultures of digested juxta-alveolar smooth muscle cells for hypoxia-induced changes. Smooth Muscle Cells (SMCs) obtained by dispase digestion of the extreme lung parenchyma were used to study the effect of CHAH in the juxta-alveolar region and foetal and maternal cells were compared. Pulmonary venous SMCs were also obtained from dissected 5th to 7th generation levels pulmonary veins (<0.5 mm). Fluorescence tagged antibodies against alpha smooth muscle actin (alpha SMA) and calponin respectively were used as markers to identify cellular structural differences by routine immunohistochemistry. Comparison of the functional integrity of the cells was made using their growth profiles obtained by radiolabeled thymidine incorporation and liquid scintillation counting. Marked differences were seen in juxta-alveolar SMCs obtained by digestion of extreme lung parenchyma of hypoxic sheep. Hypoxic adult sheep cells showed increased filamentation. Hypoxic foetal sheep cells showed internal restructuring and disorganization of both alpha-SMA and calponin filaments. The growth profiles of juxta-alveolar SMCs showed that the hypoxia-affected cells of both the foetus and adult sheep had a fast initial growth rate peaking at 48h while their normoxic equivalents had a steadier growth rate peaking at 72h. Hypoxia-affected cells showed contact inhibition at ~50% subconfluence and apoptosis by 48h. Chronic high altitude hypoxia causes both phenotypical and functional changes in pulmonary smooth muscle cells near the air/blood interface.

Letrozole increases growth of rat theca-interstitial cells and CYP17A1 gene expression in the rat ovary

Letrozole (LET) is a non-steroidal aromatase inhibitor, blocking the conversion of androgens to estrogens in granulosa cells. However, little is known regarding the effect of LET on theca-interstitial cell function. This study evaluated the effect of LET in vivo and in cultures of theca-interstitial cells. In-vitro and in-vivo studies. Effects of LET were studied in intact rats receiving either LET (90-day-continuous-release sc pellets, 400 μg/day) or placebo pellets (control group). After 10 weeks, the animals were sacrificed, ovaries were isolated and blood samples collected. In vitro experiments evaluated theca-interstitial cells isolated from 30 day-old female rats and cultured for up to 48 hrs in the absence (control) or in the presence of LET (0.1-1μM). Cell proliferation was evaluated by quantification of DNA synthesis as determined by radiolabelled thymidine incorporation assay. CYP17A1 gene expression was evaluated by qrt-PCR using HPRT as a reference gene. Androstenedione levels in plasma were determined by liquid chromatography-mass spectrometry. Comparisons between groups were performed by ANOVA followed by post-hoc pairwise testing. LET, in vivo, induced an increase in ovarian size compared to the control group: 179.1 ± 9.46 mg vs. 115 ± 5.19 mg (P<0.05). LET also induced a 57-fold (P<0.001) increase in CYP17A1 gene expression and a 52-fold (P<0.001) increase in plasma androstenedione level. In vitro, LET stimulated theca-interstitial cell proliferation in a concentration-dependent fashion by up to 65% (P<0.01) at the highest concentration (1 μM). LET stimulates growth of the theca-interstitial cell compartment and increases expression of the key enzyme (CYP17A1) regulating the androgen biosynthesis pathway in the rat. The present findings reveal novel mechanisms of action of LET in the rat ovary.

The intermediate conductance Ca2+‐activated K+ channel inhibitor TRAM‐34 stimulates proliferation of breast cancer cells via activation of oestrogen receptors

K(+) channels play a role in the proliferation of cancer cells. We have investigated the effects of specific K(+) channel inhibitors on basal and oestrogen-stimulated proliferation of breast cancer cells. Using the mammary adenocarcinoma cell line MCF-7 we assayed cell proliferation by radiolabelled thymidine incorporation in the absence or presence of various K(+) channel inhibitors with or without 17beta-oestradiol. Inhibitors of K(v)10.1 and K(Ca)3.1 K(+) channels suppressed basal proliferation of MCF-7 cells, but not oestrogen-stimulated proliferation. TRAM-34, a specific inhibitor of K(Ca)3.1 channels increased or decreased cell proliferation depending on the concentration. At intermediate concentrations (3-10 microM) TRAM-34 increased cell proliferation, whereas at higher concentrations (20-100 microM) TRAM-34 decreased cell proliferation. The enhancement of cell proliferation caused by TRAM-34 was blocked by the oestrogen receptor antagonists ICI182,780 and tamoxifen. TRAM-34 also increased progesterone receptor mRNA expression, decreased oestrogen receptor-alpha mRNA expression and reduced the binding of radiolabelled oestrogen to MCF-7 oestrogen receptor, in each case mimicking the effects of 17beta-oestradiol. Our results demonstrate that K(+) channels K(v)10.1 and K(Ca)3.1 play a role in basal, but not oestrogen-stimulated MCF-7 cell proliferation. TRAM-34, as well as inhibiting K(Ca)3.1, directly interacts with the oestrogen receptor and mimics the effects of 17beta-oestradiol on MCF-7 cell proliferation and gene modulation. Our finding that TRAM-34 is able to activate the oestrogen receptor suggests a novel action of this supposedly specific K(+) channel inhibitor and raises concerns of interpretation in its use.

In vitro activity of pyrvinium pamoate against Entamoeba histolytica and Giardia intestinalis using radiolabelled thymidine incorporation and an SYBR Green I-based fluorescence assay

To assess the in vitro activity of the FDA-approved antihelminthic drug pyrvinium pamoate against Entamoeba histolytica and Giardia intestinalis. A head-to-head comparison of a standard radiolabelled thymidine incorporation assay and the SYBR Green I-based fluorescence assay for determination of in vitro inhibition by pyrvinium and metronidazole was performed. The 50% inhibitory concentration (IC(50)) for treatment of E. histolytica with pyrvinium was 4-5 microM for both assays compared with 1-2 microM for metronidazole. For pyrvinium treatment of G. intestinalis, an IC(50) of approximately 12 microM was determined by the radiolabelled thymidine assay alone, with maximum inhibition around 60%. In contrast, the IC(50) for metronidazole treatment using this assay was approximately 2 microM. Pyrvinium is a potential gut lumen agent for treatment of intestinal amoebiasis, but possibly not for giardiasis. SYBR Green I is an alternative screening method for E. histolytica, but not G. intestinalis.

Dichloromethane extract of Dicerocaryum senecioides leaves exhibits remarkable anti-inflammatory activity in human T-lymphocytes

The leaves of Dicerocaryum senecioides are used in South Africa as a traditional remedy for many ailments, including inflammatory disorders. The purpose of this study was therefore to evaluate the anti-inflammatory potential of a dichloromethane extract of D. senecioides leaves. Methanol extracts of the leaves were sub-fractionated with dichloromethane and the anti-inflammatory potential of this fraction investigated according to its effects on the mitogen-induced proliferative responses and cytokine profiles of isolated human blood mononuclear leucocytes (MNL). The cells were pre-treated with the extract (25–100 µg mL−1) followed by addition of the mitogen, phytohaemagglutinin (PHA, 5 µg mL−1 final), and measurement of lymphocyte activation and proliferation, using flow cytometric detection of up-regulation of expression of CD25 and incorporation of radiolabelled thymidine into newly synthesised DNA, respectively. Cytokine production by unstimulated and PHA-activated cells was measured using multiplex suspension bead array technology. Treatment of cells with the Dicerocaryum extract resulted in dose-related inhibition of PHA-activated lymphocyte proliferation and expression of CD25, as well as decreased production of Th1 (IFN-γ, TNF-α) and Th2 (IL-10) cytokines. These observations not only underscore the anti-inflammatory potential of components of Dicerocaryum leaves, but also provide a basis for future definitive studies.

Changes in insulin and IGF-I receptor expression during differentiation of human preadipocytes

Mature adipocytes originate from fibroblast-like precursor cells, preadipocytes, which differentiate to obtain the characteristics of adipocytes. Our aim was to investigate how differentiation of human preadipocytes affects the distribution of insulin receptors (IR) and IGF-I receptors (IGF-IR) and other cell characteristics. Preadipocytes were differentiated using indomethacine, dexamethasone, isobutyl-methylxantine (IBMX) and high concentration of insulin. Gene expression was quantified by real-time RT-PCR in preadipocytes (PA), differentiated preadipocytes (dPA) and mature adipocytes (mAD). The amount of expressed receptor protein was analyzed using receptor specific ELISAs and Western blot. We also studied DNA synthesis with radiolabeled thymidine incorporation and glucose accumulation with radiolabeled glucose. Differentiation of PA increased gene expression of IR but not IGF-IR. GLUT4, growth hormone receptor (GHR) and adiponectin appeared or increased. In PA and dPA only IR-A was expressed whereas also IR-B was detected in mAD. By Western blot and ELISA, IR and IGF-IR were detected in PA, dPA and mAD. During differentiation the ratio of IR to IGF-IR increased severalfold. In PA both the IR and the IGF-IR was phosphorylated by their own ligand at 1 nM and in dPA the activation of both receptors was stimulated by IGF-I, but not insulin, at 1 nM. Accumulation of glucose in PA was increased by insulin at 10 nM and by IGF-I at 1 nM and 10 nM. DNA synthesis was increased by insulin and IGF-I at 10 nM. In conclusion, both IR and IGF-IR are present in human preadipocytes and adipocytes. Differentiation is characterized by an increased IR/IGF-IR ratio.

Acting via a Cell Surface Receptor, Thyroid Hormone Is a Growth Factor for Glioma Cells

Recent evidence suggests that the thyroid hormone L-thyroxine (T4) stimulates growth of cancer cells via a plasma membrane receptor on integrin alphaVbeta3. The contribution of this recently described receptor for thyroid hormone and receptor-based stimulation of cellular mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)] activity, to enhancement of cell proliferation by thyroid hormone was quantitated functionally and by immunologic means in three glioma cell lines exposed to T4. At concentrations of 1 to 100 nmol/L, T4 caused proliferation of C6, F98, and GL261 cells, measured by accumulation of proliferating cell nuclear antigen (PCNA) and radiolabeled thymidine incorporation. This effect was inhibited by the T4 analogue, tetraiodothyroacetic acid, and by an alphaVbeta3 RGD recognition site peptide, both of which block T4 binding to integrin alphaVbeta3 but are not agonists. Activation of MAPK by T4 was similarly inhibited by tetraiodothyroacetic acid and the RGD peptide. The thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) and T4 were equipotent stimulators of PCNA accumulation in C6, F98, and GL261 cells, but physiologic concentrations of T3 are 50-fold lower than those of T4. In conclusion, our studies suggest that glioblastoma cells are thyroid hormone dependent and provide a molecular basis for recent clinical observations that induction of mild hypothyroidism may improve duration of survival in glioblastoma patients. The present experiments infer a novel cell membrane receptor-mediated basis for the growth-promoting activity of thyroid hormone in such tumors and suggest new therapeutic approaches to the treatment of patients with glioblastoma.

Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin in Escherichia coli

The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

Parasympathetic nerve-evoked protein synthesis, mitotic activity and salivary secretion in the rat parotid gland and the dependence on NO-generation

Incorporation of radiolabelled leucine and thymidine into trichloroacetic acid-insoluble material of the parotid gland was used as indices of protein synthesis and mitotic activity, respectively, following electrical stimulation of the parasympathetic auriculo-temporal nerve for 30 min in pentobarbitone-anaesthetized rats under adrenoceptor blockade (phentolamine and propranolol, 2mg/kg intravenous of each) in the absence or presence of atropine (2mg/kg intravenous) and without or with nitric oxide synthase inhibitors. In atropinized rats, the parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked mean increases in protein synthesis at a frequency of 10 Hz (142%) and 40 Hz (200%) were not affected in a statistically significant way (124 and 275%, respectively) by the neuronal type NO-synthase inhibitor N(w)propyl-l-arginine (N-PLA) (30 mg/kg intravenous). Neither were the increase (175%) in protein synthesis at 10 Hz in non-atropinized animals affected by N-PLA (180%). The increase (65%) in mitotic activity, 19 h after the end of stimulation at 40 Hz, in the presence of atropine, was not affected by N-PLA (55%). Neither were the increase (95%) in gland content of amylase at this point of observation statistically significant affected by N-PLA (144%). The secretion of fluid and output of amylase from the parotid gland upon nerve stimulation was not affected by N-PLA. When examining the non-selective NO-synthase inhibitor l-NAME (30 mg/kg intravenous) in atropinized rats subjected to stimulation at 10 Hz, neither the increase in protein synthesis nor the evoked fluid response or amylase outputs were affected. Hence, in contrast to an NO-dependent sympathetic-induced protein synthesis and mitosis in the parotid gland, involving the activity of the neuronal type NO-synthase, no support for a parasympathetic-induced protein synthesis (and gain in gland amylase) and mitosis, depending on NO-generation, was found. Likewise, the present findings provide no evidence for a role of NO in the parasym pathetic nerve-evoked fluid secretion and amylase output.

Acidic Fibroblast Growth Factor (FGF-1) and FGF Receptor 1 Signaling in Human Y79 Retinoblastoma

Fibroblast growth factors (FGFs) represent potent effectors and play essential roles in both normal development and many pathological processes. Little is known about their possible implication in retinoblastoma growth. We sought to examine FGF high- and low-affinity receptor (FGFR) expression, activation of FGFR1 by acidic FGF (FGF-1), and proliferative effects on Y79 cells. Expression of FGFR1 to FGFR4 was screened in Y79 cells by means of immunochemical and reverse transcriptase polymerase chain reaction techniques. Tyrosine phosphorylation of FGFR1 induced by FGF was examined by immunoprecipitation after stimulation with FGF-1 in the presence or absence of heparin. Retinoblastoma proliferation was monitored by radiolabeled thymidine incorporation or a vital dye-based assay, after addition of FGF-1 with or without inclusion of a specific FGFR1 neutralizing antibody or FGFR1 antisense oligonucleotides. Low-affinity heparan sulfate proteoglycan coreceptors were blocked through sodium chlorate or heparinase treatment of Y79 cells. Y79 retinoblastoma expressed all 4 FGFRs, at both the protein and messenger RNA levels. The FGFR1 was differentially phosphorylated in a time- and heparin-dependent manner by FGF-1. Proliferation of Y79 cells induced by FGF-1 was entirely mediated by FGFR1, since inclusion of specific neutralizing antibodies or antisense oligonucleotides completely prevented tumor cell multiplication. Finally, FGF-1-induced proliferation was dependent on the presence and sulfation of heparan sulfate proteoglycan. Y79 retinoblastoma expresses all 4 FGFRs, but FGFR1 activation entirely accounts for FGF-1-driven cell proliferation. These studies demonstrate a role for the FGF-1/FGFR1 pathway in retinoblastoma proliferation, and may contribute to developing therapeutic strategies to limit retinoblastoma growth.

Interplay between paracrine signaling and gap junctional communication in ovarian follicles

Intercellular communication is required for ovarian folliculogenesis. This is apparent in mice lacking connexin43 (Cx43, a gap junction protein strongly expressed in granulosa cells), or growth/differentiation factor-9 (GDF9, an oocyte-specific growth factor that stimulates granulosa cell proliferation and differentiation), or in mice expressing a mutant form of Kit ligand (KITL, a paracrine factor that, in the ovary, is secreted by granulosa cells to stimulate oocyte growth). In all of these mutant lines, follicle growth is impaired suggesting a possible interaction between paracrine signaling and gap junctional communication. To assess this possibility, we analyzed gene expression in mutant ovaries. Despite the lack of gap junctional coupling between granulosa cells of Cx43 null mutant ovaries, expression of the genes encoding KITL and its receptor, KIT, is maintained. Furthermore, GDF9 expression is maintained. In GDF9 null mutant ovaries, there is no apparent change in Cx43 expression and, correspondingly, the granulosa cells remain coupled. There is also no increase in granulosa cell apoptosis in ovaries lacking Cx43 or GDF9. Staining for proliferating cell nuclear antigen (PCNA) revealed that the granulosa cells of Cx43 null mutant ovaries have a reduced frequency of DNA synthesis. Using both radiolabeled thymidine incorporation and PCNA staining in vitro, we showed that recombinant GDF9 could restore the proliferation of coupling-deficient granulosa cells to the level of control cells. These results indicate that impaired folliculogenesis in mice lacking Cx43 is due at least in part to reduced responsiveness of granulosa cells to oocyte-derived GDF9, indicating an interaction between these two modes of intercellular communication.

Troglitazone Acts on Cellular pH and DNA Synthesis through a Peroxisome Proliferator-Activated Receptor γ-Independent Mechanism in Breast Cancer-Derived Cell Lines

The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na(+)/H(+) exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation. Intracellular pH (pH(i)) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) gamma involvement was assessed using an antagonist and PPARgamma(-/-) NIH3T3 cells. TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 +/- 0.23 to 6.77 +/- 0.06; P < 0.001) and MDA-MB-231 cells (7.38 +/- 0.18 to 6.89 +/- 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH(4)Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 micromol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TRO-mediated inhibition of proliferation was not reversed by the presence of the PPARgamma inhibitor GW9662 and was demonstrable in PPARgamma(-/-) NIH3T3 cells, consistent with a PPARgamma-independent mechanism. TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPARgamma antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPARgamma.