Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.     

Activated MEK stimulates expression of AP-1 components independently of phosphatidylinositol 3-kinase (PI3-kinase) but requires a PI3-kinase signal To stimulate DNA synthesis

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.     

Acute effects of prostaglandin F2 alpha, luteinizing hormone, and estradiol on second messenger systems and on the secretion of oxytocin and progesterone from granulosa and early luteal cells of the ewe

Previous reports have suggested that gonadotropins, estradiol, and prostaglandin F2 alpha (PGF2 alpha) have varying effects on progesterone and oxytocin synthesis or secretion in cultured granulosa and luteal cells collected at different stages of the estrous cycle. The experiments reported here were designed to investigate whether effects of these agonists on secretion of hormones and their coupling to second messenger systems changed around the time of ovulation. Granulosa cells and Day 2 luteal cells of the ewe were cultured for three days and then treated for 30 min with varying doses of PGF2 alpha, LH, or estradiol. LH increased intracellular cAMP at both stages, but granulosa cells were more responsive in terms of both minimum effective dose (10 compared with 100 ng/ml) and degree of stimulation. LH caused no change in intracellular inositol phosphate levels. Both granulosa and early luteal cells responded to LH treatment by an increase in progesterone output in a dose-responsive fashion. PGF2 alpha increased inositol phosphate accumulation in cells collected at both stages of the cycle. All doses tested (10(-6)-10(-8) M) stimulated the release of oxytocin into the culture medium from both granulosa and luteal cells. Progesterone secretion was also increased, but only at the highest dose (10(-6) M). Estradiol treatment (10(-6) M) did not affect either the inositol phosphate or cAMP second messenger systems, but it did inhibit the secretion of oxytocin from granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.     

Differential inhibition by hyperammonemia of the electron transport chain enzymes in synaptosomes and non-synaptic mitochondria in ornithine transcarbamylase-deficient spf-mice: restoration by acetyl-L-carnitine

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

The influence of medium formulation on phosphomonoester and UDP-hexose levels in cultured human colon tumor cells as observed by 31P NMR spectroscopy

High-resolution 31P NMR spectroscopy at 11.7 T was used to examine the influence of medium formulation (medium and serum type, and concentrations of glucose and inositol) on the cellular phosphate metabolism of CX-1 cells, a human colon cancer cell line derived from HT-29 cells. Striking differences in the 31P spectra of harvested CX-1 cells were observed. The largest variation was seen in the phosphocholine and UDP-hexose levels (up to seven-fold changes), with smaller differences in the levels of other phosphate metabolites. The major UDP-hexose species were found to be UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (ca 2:1 ratio), which have been proposed in the literature to be markers of cell differentiation status. Medium-induced alterations in metabolite levels were much greater than the normal variations seen in CX-1 control samples grown under identical conditions. They even exceeded the characteristic differences observed between different human tumor cell lines grown under one set of culture conditions. The remarkable sensitivity of CX-1 cellular phosphate metabolism to the culture environment has implications for the comparison of in vitro vs in vivo spectra, and for the interpretation of effects due to growth and therapy.     

Autocrine-paracrine effects of overexpression of hepatocyte growth factor gene on growth of endothelial cells

Although hepatocyte growth factor (HGF) is synthesized in vascular cells, it is not known whether locally synthesized HGF acts similarly to exogenously added HGF. Therefore, we transfected cultured cells with human HGF vector and examined the effects on growth of vascular cells. Endothelial cells (EC) transfected with HGF vector synthesized and secreted high levels of HGF, and also showed significantly higher number. Addition of conditioned medium from vascular smooth muscle cells (VSMC) or EC transfected with HGF vector to nontransfected EC resulted in a significant increase in cell number, which was abolished by anti-HGF antibody. Co-culture of HGF-transfected VSMC with EC showed that HGF released from VSMC or EC stimulated EC growth. These results demonstrate that endogenously produced HGF by transfection of human HGF vector can exert autocrine and paracrine stimulatory effects on EC growth, but not VSMC growth, suggesting the role of local HGF system in cardiovascular disease.     

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

Mutational analysis of cis-acting sequences in the 3'- and 5'-untranslated regions of RNA2 of red clover necrotic mosaic virus

BACKGROUND/AIMS: Hepatic stellate cells appear to be the main producers of hepatocyte growth factor of the normal liver. Insulin-like growth factors in doses over 20 ng/ml have been reported to stimulate hepatocyte growth factor production in cultured hepatic stellate cells. The aim of the present study was to investigate whether parenchymal cell conditioned medium had insulin-like growth factor-independent effects on hepatic stellate cells. METHODS: Primary rat hepatic stellate cells were cultured for 1-7 days. DNA synthesis was measured by 3H-thymidine incorporation. Hepatocyte growth factor and transforming growth factor beta1 immunoreactivity was quantified by ELISA. Hepatocyte growth factor mRNA levels were determined with gel RNase protection assay. Parenchymal cell conditioned medium was obtained from hepatocytes cultured for 2 days in medium without added serum or hormones. RESULTS: Incubation of 1-7-day-old hepatic stellate cells for 2 days with parenchymal cell conditioned medium enhanced the medium content of hepatocyte growth factor. Parenchymal cell conditioned medium contained less than 5.0 ng/ml immunoreactive insulin-like growth factor-1 as measured by radio immunoassay. Parenchymal cell conditioned medium did not contain any insulin-like growth factor bioactivity measured as phosphorylation of type 1 insulin-like growth factor receptor beta subunit and a protein with a size consistent with that of insulin receptor substrate-1. The stimulatory effect of parenchymal cell conditioned medium on hepatocyte growth factor was time- and dose-dependent. The effects of a high dose of parenchymal cell conditioned medium (dilution 1:2 containing less than 2.5 ng/ml insulin-like growth factor-1) were additive to that of high doses (100 ng/ml) of insulin-like growth factor-1 or des (1-3) insulin-like growth factor-1, an analogue with low affinity to insulin-like growth factor binding proteins. Neither parenchymal cell conditioned medium nor insulin-like growth factor-1 enhanced transforming growth factor beta1 immunoreactivity in the medium. Both parenchymal cell conditioned medium and insulin-like growth factor-1 stimulated DNA synthesis in hepatic stellate cells, confirming previous reports. CONCLUSIONS: The present results indicate that both insulin-like growth factor-1 and insulin-like growth factor-1-independent factors from hepatocytes can stimulate hepatocyte growth factor production by hepatic stellate cells. Therefore, insulin-like growth factor-1 and other hepatocyte-derived factors may indirectly affect hepatocytes via a paracrine loop.     

Human androgen-induced growth factor in prostate and breast cancer cells: its molecular cloning and growth properties

Androgen-induced growth factor (AIGF) has hormone-regulated properties in the mouse Shionogi carcinoma cell line. To investigate whether or not it is involved in growth of human hormone-responsive cancers, we isolated the human AIGF gene from a placental genomic library. Genomic analyses suggested that the AIGF gene was about 6.5 kilobases in length containing five exons. The deduced amino acid sequence of human AIGF was completely identical with that of the mouse. RT-PCR analyses showed that prostate and breast cancer cell lines, LNCaP, PC-3, and MCF-7, slightly expressed the AIGF gene. Recombinant AIGF enhanced the growth of the human prostate cancer LNCaP cells, and it also markedly stimulated the growth of fibroblasts. These in vitro findings suggest that AIGF might be a possible autocrine or paracrine factor in hormone-responsive cancers.     

Evidence for differential regulation of calcium by outer versus inner hair cells: plasma membrane Ca-ATPase gene expression

The expression of mRNA encoding plasma membrane calcium ATPase (PMCA) subunit isoforms (1-4) and splice variants was examined in the adult and developing rat cochlea by PCR and in situ hybridization. High levels of PMCA mRNA expression were observed in the neurons of the spiral ganglion, and in hair cells. Spiral ganglion neurons expressed PMCA 1-3 beginning in embryonic development, reaching high levels shortly after birth, and continuing into adulthood. Inner hair cells expressed PMCA 1 at moderate levels from birth to the time of onset of cochlear function on postnatal day 12, and strongly from then until adulthood. Outer hair cells expressed PMCA 2 at high levels from shortly after birth through adulthood. The data suggest that the calcium clearance requirements of inner and outer hair cells are distinct. PMCA 2 is the isoform with the highest affinity for calmodulin, and has also been associated with high levels of inositol triphosphate. Its presence in outer hair cells suggests that regulation of the enzyme by calmodulin may be particularly important for this hair cell type. It further suggests that inositol phosphate may play a unique role in the outer hair cell.     

Pathophysiological role of the cytokine network in the anterior pituitary gland

Recent evidence has demonstrated that cytokines and other growth factors act in the anterior pituitary gland. Using the traditional criteria employed to determine autocrine or paracrine functions our review shows that, in addition to their role as lymphocyte messengers, certain cytokines are autocrine or paracrine regulators of anterior pituitary function and growth. The cytokines known to regulate and/or be expressed in the anterior pituitary include the inflammatory cytokine family (IL-1 and its endogenous antagonist, IL-1ra; TNF-alpha, and IL-6), the Th1-cytokines (IL-2 and IFN-gamma), and other cytokines such as LIF, MIF, and TGF-beta. This review examines at the cellular, molecular, and physiological levels whether: (1) each cytokine alters some aspect of pituitary physiology; (2) receptors for the cytokine are expressed in the gland; and (3) the cytokine is produced in the anterior pituitary. Should physiological stimuli regulate pituitary cytokine production, this would constitute additional proof of their autocrine/paracrine role. In this context, we analyze in this review the current literature on the actions of cytokines known to regulate anterior pituitary hormone secretion, selecting the in vivo studies that support the direct action of the cytokine in the anterior pituitary. Further support for direct regulatory action is provided by in vitro studies, in explant cultures or pituitary cell lines. The cytokine receptors that have been demonstrated in the pituitary of several species are also discussed. The endogenous production of the homologous cytokines and the regulation of this expression are analyzed. The evidence indicating that cytokines also regulate the growth and proliferation of pituitary cells is reviewed. This action is particularly important since it suggests that intrinsically produced cytokines may play a role in the pathogenesis of pituitary adenomas. The complex cell to cell communication involved in the action of these factors is discussed.     

Dislodgment and accelerated degradation of Ras

Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.     

Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor)

The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.     

Oncogenic Ha-Ras-dependent mitogen-activated protein kinase activity requires signaling through the epidermal growth factor receptor

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.     

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.     

Excessive activation of tyrosine kinases leads to inhibition of proliferation in a thyroid carcinoma cell line

Autocrine stimulation of growth is a hallmark of many tumor cell lines. In this work we investigated the synthesis and secretion of growth factors and the expression of their corresponding receptors in HTC-TSHr thyroid carcinoma cells. These cells synthesize epidermal growth factor (EGF) receptors and platelet-derived growth factor beta (PDGF beta) receptors and in addition transforming growth factor alpha (TGF alpha), PDGF-A and PDGF-B chains, respectively. Addition of EGF or PDGF-BB to the culture medium resulted in growth inhibition of HTC-TSHr cells. In contrast, treatment of the cells with low concentrations of neutralizing anti-TGF alpha antibodies or tyrosine kinase inhibitors led to stimulation of cell proliferation. Low concentrations of neutralizing anti-PDGF-B antibodies did not affect growth of the cells. As expected, cell proliferation was inhibited when high concentrations of either neutralizing anti-TGF alpha antibodies or anti-PDGF-B antibodies were applied. PDGF-AA did not influence growth of HTC-TSHr cells. We conclude that growth of HTC-TSHr thyroid carcinoma cells is influenced by two autocrine loops between TGF alpha and EGF receptors and between PDGF-B and PDGF beta receptors. However, our data suggest that excessive activation of tyrosine kinase receptors in these cells results in a relative inhibition rather than stimulation of growth.