The role of mitogen-activated protein kinase and protein kinase C in fibronectin production in human vascular smooth muscle cells.

BACKGROUND: After evaluating various growth factors, cytokines, and extracellular matrix (ECM) proteins, we found that the most potent agonists of smooth muscle cell (SMC) fibronectin (Fn) production were transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF). To determine the possible signaling pathways involved in the production of this matrix protein, we investigated the role of the intracellular proteins, protein kinase C (PKC) and mitogen-activated protein kinase (MAP-K), in TGF-beta- and EGF-induced human vascular SMC Fn production. MATERIALS AND METHODS: After stimulation of human SMCs with TGF-beta (10 ng/ml) and EGF (100 ng/ml), Fn in the cell medium was assayed by immunoblotting using a specific antibody. PKC was activated by brief stimulation of SMC with phorbol 12,13-dibutyrate (PDBu) and inhibited by downregulation with PDBu or the inhibitor, GF109203X. MAP-K was inhibited with PD098059. RESULTS: PKC activation increased basal and synergistically enhanced TGF-beta- and EGF-induced Fn production. However, inhibition of PKC by downregulation and GF109203X did not diminish Fn production by TGF-beta and EGF. Surprisingly, these two methods of inhibition slightly increased basal and agonist-induced Fn production. The MAP-K kinase inhibitor, PD098059, produced an almost complete inhibition of EGF and a partial inhibition of TGF-beta-induced Fn production. CONCLUSIONS: Activation of PKC stimulates Fn production; however, neither TGF-beta nor EGF produce Fn through a PKC-dependent pathway. EGF and TGF-beta both stimulate Fn production at least in part through the intracellular signaling protein MAP-K. Understanding the signaling pathways involved in extracellular matrix protein production will allow the design of specific inhibitors of intimal hyperplasia.     

Protein kinase C and calcium regulation of adenylyl cyclase in isolated rat pancreatic islets.

Rat islets express several isoforms of adenylyl cyclase (AC), and the regulation of AC activity in isolated islets by Ca(2+) and protein kinase C (PKC) was investigated. At basal 2.8 mmol/l glucose, the muscarinic receptor agonist carbamylcholine chloride (CCh) evoked a concentration-dependent increase in cAMP generation with a maximum increase at least 4.5-fold above control. In contrast, forskolin and glucagon-like peptide 1 fragment 7-36 amide increased cAMP accumulation 23-fold and almost 10-fold, respectively. Cholecystokinin 26-33 sulfated amide (CCK) also stimulated cAMP production by up to eightfold, as did the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu and CCh or CCK responses were not additive. The effects of phorbol ester, CCh, and CCK were inhibited by as much as 75% by the PKC inhibitors GF 109203X and Ro-32-0432 and after PKC downregulation. In the absence of extracellular Ca(2+), PDBu-, CCh-, and CCK-induced cAMP production was inhibited by approximately 50% in each case. Chelation of intracellular Ca(2+) with 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) inhibited CCh- and CCK-stimulated cAMP generation by approximately 50% but did not inhibit the stimulatory effect of PDBu. Stringent Ca(2+) depletion by removal of extracellular Ca(2+) and inclusion of BAPTA/AM allowed for increased cAMP production in response to CCh and CCK; PKC inhibitors and PKC downregulation prevented this stimulation. Glucose stimulation also increased islet cAMP production, but PDBu did not potentiate the glucose response. The results suggest that Ca(2+) influx, Ca(2+) mobilization, and PKC activation play important roles in the modulation of AC activity in pancreatic islets.     

Inhibition of growth of established human glioma cell lines by modulators of the protein kinase-C system

The protein kinase-C (PKC) second messenger system contributes to regulation of cell growth and differentiation. This study was undertaken to examine the effects of modulators of the PKC enzyme system on the state of differentiation and proliferation rates of human gliomas in vitro. The administration of the PKC-activating phorbol esters 4-beta-phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) resulted in a dose-related inhibition of growth of human glioma cell lines in vitro as measured by 3H-thymidine uptake. The synthetic nonphorbol PKC activator (SC-9) produced an even more pronounced decrease of 3H-thymidine uptake. Diacylglycerol, an endogenous activator of the system, applied externally, transiently decreased the proliferation, in concordance with its short-lived existence in vivo. Conversely, the administration of 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), a phorbol ester that binds but does not activate the enzyme, had no effect on the proliferation rate. At the dosages that maximally decreased proliferation, there was no evidence of direct glioma cell lysis induced by these agents as measured by a chromium-release assay. Immunocytochemical analysis and cytofluorometric measurement of glial fibrillary acidic protein (GFAP) staining in the treated cultures revealed an increase in GFAP staining over control cultures. In contrast to the response of glioma cells, nonmalignant human adult astrocytes treated with the PKC activators responded by increasing their proliferation rate. The authors postulate that the diametrically opposed effects of PKC activators on nonmalignant astrocytes versus glioma growth may be due to a high intrinsic PKC activity in glioma cells, with resultant down-regulation of enzyme activity following the administration of the pharmacological activators.     

Phorbol esters modulate the high Ca2(+)-stimulated accumulation of inositol phosphates in bovine parathyroid cells

We examined the effects of TPA on the high Ca2(+)-stimulated accumulation of inositol phosphates in bovine parathyroid cells to determine whether protein kinase C modulates phosphoinositide turnover in a fashion similar to that observed in other cell types stimulated by more classic Ca2+ mobilizing hormones. Following exposure of parathyroid cells to TPA (10(-6) M) for 10 or 30 minutes, there was a time- and dose-dependent inhibition of the accumulation of inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) stimulated by 3 mM Ca2+. Half the maximal observed inhibition took place at 1-10 nM TPA, with 50-60% inhibition of high Ca2(+)-stimulated accumulation of inositol phosphates at 10(-6) M TPA. The active phorbol ester, 4 beta-phorbol didecanoate, produced similar effects; the inactive derivative, 4 alpha-phorbol didecanoate, was without effect. When parathyroid cells were exposed to TPA (10(-6) M) for varying times and were then incubated with high (3 mM) Ca2+, inhibition of inositol phosphate accumulation was observed with 10 or 30 minutes preincubation. In contrast, preincubation of cells with TPA for 3 or 18 h markedly enhanced the high (3 mM) Ca2(+)-induced increase in inositol phosphates. In cells preincubated with TPA for 18 h, binding sites for [3H]phorbol dibutyrate and total protein kinase C (PKC) activity were reduced by greater than 95% and by 71%, respectively, consistent with downregulation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Propofol inhibits phorbol 12, 13-dibutyrate-induced, protein kinase C-mediated contraction of rat aortic smooth muscle

BACKGROUND: Propofol induces dose-dependent vasodilation and hypotension in the clinical situation, and protein kinase C (PKC)-mediated Ca2+ sensitization plays an important role in vascular smooth muscle contraction. This study is designed to examine the effects of propofol on the active phorbol ester (phorbol 12, 13-dibutyrate; PDBu)-induced, PKC-mediated contraction of rat aortic smooth muscle. METHODS: The PDBu-induced contraction of endothelium-denuded rat aortic rings was measured in the presence or absence of PKC inhibitor, bisindolylmaleimide I, or propofol, using isometric force transducers. The PDBu-induced PKC phosphorylation of endothelium-denuded rat aortic strips was detected in the presence or absence of bisindolylmaleimide I or propofol, using Western blotting. RESULTS: PDBu, but not the inactive phorbol ester, 4-alpha-phorbol 12-myristate-13-acetate, dose-dependently induced both a slowly developing sustained contraction and PKC phosphorylation of rat aortic smooth muscle, reaching the peak level at the concentration of 10(-6) M. The PDBu (10(-6) M)-induced contraction was dose-dependently inhibited by bisindolylmaleimide I with reductions of 6.8 +/- 1.8% (P > 0.05), 39.8 +/- 8.7% (P < 0.01) and 96.7 +/- 1.4% (P < 0.01) in response to concentrations of 5 x 10(-7) M, 10(-6)x M and 5 x 10(-6) M, respectively, and by propofol with decreases of 5.2 +/- 1. 6% (P > 0.05), 9.4 +/- 1.7% (P < 0.05), 65.3 +/- 9.2% (P < 0.01) and 96.2 +/- 1.6% (P < 0.01) in response to concentrations of 5 x 10(-7) M, 10(-6) M, 5 x 10(-6) M and 10(-5) M, respectively. Both bisindolylmaleimide I and propofol also inhibited the PDBu-induced increase in the density of the phosphorylated PKC bands in a dose-dependent manner, with decreases of 6.3 +/- 2.8% (P > 0.05), 42.9 +/- 3.2% (P < 0.01) and 96.6 +/- 3.4% (P < 0.01) in response to 5 x 10(-7) M, 10(-6) M or 5 x 10(-6) M bisindolylmaleimide I, respectively, and with decreases of 4.2 +/- 2.5% (P > 0.05), 13.5 +/- 1.7% (P < 0.05), 69.5 +/- 3.5% (P < 0.01) and 95.3 +/- 4.3% (P < 0.01) in response to 5 x 10(-7) M, 10(-6) M, 5 x 10(-6) M and 10(-5) M propofol, respectively. CONCLUSION: Propofol dose-dependently inhibits PDBu-induced, PKC-mediated contraction of rat aortic smooth muscle.     

Phorbol dibutyrate plus ionomycin improves the generation of cytotoxic T cells from draining lymph nodes of patients with advanced head and neck cancer.

Fifty-one cervical nodes from 19 patients with advanced head and neck cancer were stimulated with phorbol dibutyrate and ionomycin (PDBu + Io) to determine the effect of such stimulation on the generation of cytotoxic T cells and whether this stimulation could bypass the need for autologous tumor stimulation. Lymphocytes stimulated with PDBu + Io demonstrated a sixfold greater in vitro expansion and significantly increased DNA synthesis. Whereas fresh lymphocytes displayed no cytotoxicity, stimulation with PDBu + Io and culture in interleukin-2 (IL-2) led to significant cytotoxicity equivalent to that of lymphocytes stimulated with autologous tumor and IL-2. T cells with the greatest cytotoxicity were generated from patients with nodal metastases. In patients with stage IV tumors, effector cells demonstrating greater lysis of natural killer-resistant targets (Daudi cells) were associated with higher rates of recurrence (50% versus 12%, respectively, p < 0.001). Stimulation with PDBu + Io augments growth and proliferation of lymphocytes from draining lymph nodes and preserves cytotoxicity without the need for autologous tumor. Excluding the need for antigenic stimulation by autologous tumor may prove useful in adoptive immunotherapy procedures.     

Halothane Attenuation of Calcium Sensitivity in Airway Smooth Muscle: Mechanisms of Action during Muscarinic Receptor Stimulation

Background: In airway smooth muscle, muscarinic receptor stimulation is thought to increase calcium (Ca2+) sensitivity via a guanosine 5'-triphosphate (GTP)-binding protein/protein kinase C (PKC)-mediated mechanism. This study tested the hypothesis that halothane reduces Ca2+ sensitivity during muscarinic receptor stimulation by inhibiting these second messenger pathways.

Methods: A beta-escin permeabilized canine tracheal smooth muscle preparation was used in which the cytosolic Ca2+ concentration ([Ca sup 2+]i) is controlled and the GTP-binding protein/PKC pathways remain intact and can be activated. The muscarinic receptor was activated with acetylcholine plus GTP; the GTP-binding proteins were directly activated with a nonhydrolyzable form of GTP, guanosine 5'-O-(3-thiotriphosphate; GTP gamma S); and PKC was directly activated with the PKC agonist phorbol 12,13-dibutyrate (PDBu).

Results: Free Ca2+ caused a concentration-dependent increase in force. Acetylcholine plus GTP significantly decreased the median effective concentration for free Ca2+ from 0.52 +/- 0.06 micro Meter to 0.21 +/- 0.02 micro Meter, demonstrating an increase in Ca2+ sensitivity. Halothane (0.99 +/- 0.04 mM, equivalent to approximately 4 minimum alveolar concentration in dogs) significantly attenuated this increase in Ca2+ sensitivity induced by acetylcholine plus GTP, increasing the median effective concentration for free Ca2+ from 0.21 +/- 0.02 micro Meter to 0.31 +/- 0.03 micro Meter. However, halothane did not affect the increases in Ca2+ sensitivity induced by GTP gamma S or PDBu.     

Receptor‐induced phasic activity of newborn mouse bladders is inhibited by protein kinase C and involves T‐type Ca2+ channels

OBJECTIVE

To investigate the mechanisms involved in the phasic contractile activity after muscarinic receptor activation of newborn urinary bladders and to compare neonatal and adult bladders.

MATERIALS AND METHODS

Detrusor muscle strips were isolated from newborn mice (aged 0–2 days) and compared with preparations from adult mice (aged 10–12 weeks). The effects of an activator (phorbol 12,13‐dibutyrate, PDBu) and an inhibitor (GF109203X) of protein kinase C (PKC) on contractions were investigated. T‐type Ca²⁺ channels were blocked with NiCl₂.

RESULTS

The newborn urinary bladders responded with prominent phasic contractile activity in response to carbachol (1 µm ). GF109203X (3 µm ) reduced carbachol‐induced force by ≈60% in the newborn, compared with 30% in the adult. PDBu (1 µm ) enhanced the muscarinic receptor‐mediated contraction in adult bladder muscle, whereas it completely abolished the responses in the newborn. There was no inhibition after activation with depolarization (high‐K⁺) or purinergic agonists (ATP, α,β‐methylene ATP). NiCl₂ (>30 µm ) inhibited the peak responses to carbachol in the newborn and at 300 µm it completely abolished the phasic contractile response. The responses of the adult bladder muscle were only marginally affected by NiCl₂.

CONCLUSIONS

Muscarinic receptor stimulation recruits the PKC signalling pathway in both the adult and neonatal urinary bladder. Potent PKC activation is inhibitory on carbachol‐induced activation in the newborn and stimulatory in the adult bladder. Furthermore, muscarinic receptor stimulation activates T‐type Ca²⁺ channels in the newborn, but not the adult bladder.     

Response of malignant glioma cell lines to activation and inhibition of protein kinase C-mediated pathways

To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and C6), and two low-passage human glioma lines (obtained from surgical specimens) were studied. With the exception of the C6 line, all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma. At optimal concentrations (5 to 10 ng/ml), TPA produced a two- to five-fold increase in the rate of DNA synthesis (p less than 0.05) as assessed by incorporation of 3H-thymidine. However, TPA had no additive effect on the mitogenic response produced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). Inhibition of PKC using the antibiotic polymyxin B (20 micrograms/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.     

Time-dependent effects of parathyroid hormone and prostaglandin E2 on DNA synthesis by periosteal cells from embryonic chick calvaria

Both PGE₂ and PTH (1-34) caused a time- and concentration-dependent stimulation of proliferation by embryonic chick periosteal cells. Cells were exposed to the agents for different periods of time, the medium was replaced with fresh medium, and ³H-TdR incorporation was measured after 16 hours. Challenge with 10^-6 M prostaglandin E₂ (PGE₂) or 10^-7 M parathyroid hormone (1-34) (PTH) for 5 minutes produced 4- and 5.5-fold increases in ³H-TdR incorporation, respectively. Longer exposures, however, produced diminishing responses and after 45 minutes, only minimal effects or slight inhibitions were seen. These timedependent effects were also seen with forskolin and dibutyryl-cAMP; TPA on the other hand stimulated DNA synthesis after both short- and long-term exposure. Both PGE₂ and PTH (1-34) stimulated cAMP synthesis in periosteal cells but neither could be shown to stimulate protein kinase-C (PKC) at concentrations required for stimulation of proliferation, and dibutyryl-cyclic AMP (cAMP) effectively inhibited endogenous PKC activity. It is possible that the stimulation of proliferation by short-term exposure to PGE₂ and PTH (1-34) is mediated by cAMP and that the time dependency possibly stems from the inhibition of endogenous PKC activity by increased intracellular cAMP levels.     

Enhanced protein kinase C activity correlates with the growth rate of malignant gliomas in vitro.

Direct measurement of protein kinase C (PKC) activity in vitro revealed a significant increase in the activity of the enzyme in all human malignant glioma lines examined and the rat C6 tumor in comparison with control nonneoplastic astrocyte and mixed glial cultures. The total and particulate PKC activity in these cell types correlated strongly [r = 0.98 (P less than 0.001) and 0.94 (P = 0.002), respectively] with the maximal growth rates as measured by 3H-thymidine incorporation in each of the samples. An alteration in the growth rate of an individual glioma line (A172) by varying the serum concentration in the growth medium produced comparative changes in the measured PKC activity. The addition of the phorbol ester phorbol-12-myristate-13-acetate to this tumor line under high serum conditions produced down-regulation of the enzyme, which was accompanied by a corresponding reduction in thymidine incorporation. The administration of the PKC inhibitor staurosporine produced a dose-related decrease in the basal proliferation rate of glioma lines A172 and C6, as measured by 3H-thymidine uptake and confirmed by flow cytometry, indicating that the high intrinsic PKC activity is amenable to pharmacological manipulation. Cytofluorometric deoxyribonucleic acid cell cycle analysis of the tumors treated with PKC modulators demonstrated that reduced proliferation rates were caused by an inhibition of entrance into the deoxyribonucleic acid synthesis (S) phase (decrease in proliferative index), supporting the evidence that these modulators are not slowing the tumor growth in a nonspecific cytotoxic manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperphosphorylation of Na-pump contributes to defective renal dopamine response in old rats

Dopamine D1-like receptor activation causes phosphorylation and inhibition of Na,K-ATPase (Na-pump) activity in the proximal tubules, which is associated with an increase in sodium excretion. It has been shown that dopamine and SKF 38393, a D1-like receptor agonist, caused inhibition of Na,K-ATPase activity in the proximal tubules of adult (6 mo) but not of old (24 mo) Fischer 344 rats. The present study demonstrated that SKF 38393 and PDBu, a phorbol ester and protein kinase C (PKC) activator, increased phosphorylation of the alpha(1)-subunit of Na,K-ATPase in adult but not in old rats. In adult rats, SKF 38393-mediated phosphorylation was antagonized by SCH 23390, a D1-like receptor antagonist. Similarly, Na,K-ATPase activity was inhibited by SKF 38393 and PDBu in adult but not in old rats. The basal activity of Na,K-ATPase was decreased and the basal phosphorylation state of the enzyme was increased in old compared with adult rats. Basal activity of PKC was higher in old compared with adult rats, and SKF 38393 and PDBu stimulated PKC activity in adult but not in old rats. The conclusion is that the failure of D1-like receptor agonist and phorbol ester to stimulate PKC and inhibit Na,K-ATPase activity in old rats is due, at least in part, to the higher basal PKC activity and Na,K-ATPase phosphorylation in old compared with adult rats.     

Cyclosporine effect on anti-CD3 monoclonal antibody-stimulated mitogenesis, phorbol ester comitogenesis, and PGE2 production

Human peripheral blood mononuclear cells (H-PBMC) from 10 healthy donors were stimulated to proliferate with phytohemagglutinin lectin (PHA), anti-CD3 monoclonal antibody (mAb), and anti-CD3 mAb plus phorbol 12, myristate 13 acetate (TPA), a protein kinase C (PKC) agonist. Anti-CD3 mAb-mediated mitogenesis was 35-75% of that observed with PHA. When TPA was added to a dose of mAb that by itself did not cause mitogenesis, proliferation equal to 50-90% of the maximally mitogenic dose occurred. TPA did not enhance proliferation with maximally mitogenic doses of antibody. Dimethyl-prostaglandin E2, dibutyryl cyclic AMP, and forskolin (an adenyl cyclase agonist) inhibited PHA, anti-CD3, and anti-CD3/PMA-mediated mitogenesis. Cyclosporine (CSA) inhibited anti-CD3 and anti-CD3/TPA mitogenesis in a dose-dependent fashion. While CSA inhibited anti-CD3 and anti-CD3/TPA mitogenic signals, it did not affect PGE2 production by anti-CD3 mAb-stimulated H-PBMC. In the presence of CSA, PGE2 production in PHA-stimulated H-PBMC was increased. PGE2 inhibits lymphocyte proliferation via a cyclic AMP-mediated mechanism and may enhance maturation of suppressor cells. CSA inhibits anti-CD3 mAb and anti-CD3/TPA proliferative signals in H-PBMC yet has no effect or may even enhance production of suppressive PGE2. The maturation of antigen-specific suppressor cells elicited by CSA may involve active down-regulation of CD3 receptor and PKC-dependent events while PGE2 production continues.     

Wachstum und invasion beim differenzierten schilddrüsenkarzinom: stellenwert der signaltransduktion

Zusammenfassung Der Stellenwert der Signaltransduktionssysteme Adenylatzyklase, Proteinkinase C (PKC) und Tyrosinkinase für Wachstum und Invasion einer follikulären (FTC 133) und einer papillären Schilddrüsenkarzinomzellinie (PTC-UC1) wurde analysiert. Agonisten und Antagonisten von cAMP waren ohne Effekt. Der PKC-Agonist TPA stimulierte Wachstum und Invasion von FTC133 um jeweils 15%, wohingegen der PKC-Antagonist Staurosporin Wachstum und Invasion von FTC 133 um 47 bzw. 32% hemmte. Staurosporin neutralisierte den stimulierenden Effekt von Thyreotropin (TSH), nicht aber von Epidermal growth factor (EGF). Der stimulierende Effekt von EGF auf Wachstum und Invasion beider Zellinien wurde durch einen monoklonalen Antikörper gegen den EGF-Rezeptor neutralisiert. Der Tyrosinkinaseantagonist Genistein hob die EGF-, nicht aber die TSH-Stimulation auf. Pertussistoxin hemmte Zellwachstum (FTC 133: 22%) und -invasion (FTC133: 18%). Choleratoxin hatte einen weniger inhibierenden Effekt. Die Signaltransduktion von Wachstum und Invasion differenzierter Schilddrüsenkarzinome in vitro ist komplex und wird nicht allein von der Adenylatzyklase kontrolliert.

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Growth and invasion in differentiated thyroid cancer: relative value of signal transduction

We investigated the role of three different signal transduction systems adenylate-cyclase (AC), protein kinase C (PKC) and tyrosine kinase (TK) for growth and invasion of a human follicular (FTC 133) and a human papillary thyroid cancer cell line (PTC-UCI). Cyclic AMP stimulators and inhibitors had no effect at any concentration. The PKC agonist TPA enhanced both growth and invasion of FTC133 by 15%, whereas staurosporine, a PKC antagonist, inhibited growth by 47% and invasion by 32%. The latter also reversed thyrotropin (TSH) stimulation, but not epidermal growth factor (EFG) stimulation. EGF-stimulated growth and invasion of both cell lines were abolished by EGF-receptor antagonism using a monoclonal antibody. The tyrosine kinase antagonist genistein reversed EGF, but not TSH, stimulation. Pertussis toxin inhibited growth (FTC133: 22%) and invasion (FTC133: 18%). Cholera toxin was less inhibitory. Obviously, signal transduction of differentiated thyroid cancer is complex and systems other than adenylate cyclase are crucial for basal invasion and growth of follicular thyroid cancer cells in culture.

Die Arbeit wurde durch ein Stipendium der Deutschen Forschungsgemeinschaft (DFG) (Ho 1191/2-1) gefördert     

Growth inhibition of androgen-insensitive human prostate carcinoma cells by a 19-norsteroid derivative agent,mifepristone

Mifepristone, also known as RU 486, is a 19-norsteroid derivative. Currently, mifepristone is being tested in clinical trials on meningioma and breast cancer. In this study we analyzed whether mifepristone could inhibit the growth of human prostate cancer cells including androgen-insensitive (PC-3 and DU145) and androgen-sensitive (LNCaP) cell lines. At 1-nM concentration, mifepristone exhibited a marginal stimulatory action on LNCaP and PC-3 cells. Nevertheless, a dose-dependent growth inhibition on those same cell lines was observed at concentrations of 1 μM and 10 μM. Twenty-day exposure to the clinically achievable concentration of 1 μM mifepristone resulted in consistent inhibition of all three cell lines studied. Furthermore, this in vitro growth inhibition was reflected in an in vivo nude mouse system. Mifepristone at the dosage of 4 mg/100 g body weight completely suppressed the growth of PC-3 tumors for 21 days, although this was followed by a growth rate similar to that of the control tumor. To understand the possible mechanism of mifepristone inhibition, PC-3 cells were exposed to mifepristone in comparison with dexamethasone (Dex), progesterone, and 5 alpha-dihydrotestosterone (DHT), each at 1-μM concentration. The results demonstrated that while both DHT and Dex alone had essentially no effect on cell growth, progesterone alone resulted in a 20% growth inhibition, while mifepristone had more than 60% inhibition with a 16-day exposure. At an equal concentration, the degree of growth inhibition of PC-3 cells by mifepristone or progesterone was partially diminished by simultaneous exposure to Dex. In conclusion, our results demonstrated that the growth of androgen-insensitive prostate cancer cells can be directly inhibited by mifepristone in cultures. This in vitro inhibition is reflected in xenografted tumors.     

Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK

BACKGROUND: We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth. METHODS: Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed. RESULTS: Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth. CONCLUSIONS: These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.     

Effects of suramin on the proliferation of primary epithelial cell cultures derived from normal,benign hyperplastic and cancerous human prostates

Primary epithelial cultures (PECs) derived from normal, benign hyperplastic (BPH), and cancerous human prostate tissue were treated with increasing doses of suramin, and assayed for cell proliferation over a period of days. The suramin IC₅₀ (inhibitory concentration 50%) value was 0.5 to 1.0 × 10^?4 M whereas doses between 2.5 × 10^?4 and 5 × 10^?4 M resulted in total growth inhibition. This inhibition was reversible by exchange with suramin-free medium up to day 6. Concentrations ? 5 × 10^?4 M resulted in increased cytotoxicity as exposure time increased. No differential response to suramin could be demonstrated among the prostate PECs derived from different tissues. The established cell lines, PC-3 and DU 145, grown in serum containing medium exhibited IC₅₀s comparable to the PECs grown in serum free medium. EGF, bFGF, α, or β ECGF at the concentrations tested did not reverse suramin inhibition. Increasing concentrations of bovine pituitary extract (BPE) increased cell growth in both the treated and the control cells. However, the percent growth inhibition by suramin at each concentration of BPE remained constant. Flow cytometry examination of cells treated for 7 days with suramin (0–10^?3 M) failed to detect any significant cell cycle alterations compared to control. At high concentrations of suramin (? 10^?4 M), large numbers of viable and dead cells were detectable in the medium. The increase in unattached viable cells was most prevalent (80%) in cultures treated with suramin at the time of plating, but also occurred with cells (25–30%) plated hours prior to the addition of suramin. Treatment for several days with low concentrations of suramin (10^?7 to 10^?5 M) transiently enhanced cell growth compared to Controls. © 1993 Wiley-Liss. Inc.     

Irinotecan: a potential new chemotherapeutic agent for atypical or malignant meningiomas

OBJECT: There is currently no effective chemotherapy for meningiomas. Although most meningiomas are treated surgically, atypical or malignant meningiomas and surgically inaccessible meningiomas may not be removed completely. The authors have investigated the effects of the topoisomerase I inhibitor irinotecan (CPT-11) on primary meningioma cultures and a malignant meningioma cell line in vitro and in vivo. METHODS: The effects of irinotecan on cellular proliferation in primary meningioma cultures and the IOMM-Lee malignant meningioma cell line were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. Apoptosis following drug treatment was evaluated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and the DNA laddering assays. The effects of irinotecan in vivo on a meningioma model were determined with a subcutaneous murine tumor model using the IOMM-Lee cell line. Irinotecan induced a dose-dependent antiproliferative effect with subsequent apoptosis in the primary meningioma cultures (at doses up to 100 microM) as well as in the IOMM-Lee human malignant meningioma cell line (at doses up to 20 microM) irinotecan. In the animal model, irinotecan treatment led to a statistically significant decrease in tumor growth that was accompanied by a decrease in Bcl-2 and survivin levels and an increase in apoptotic cell death. CONCLUSIONS: Irinotecan demonstrated growth-inhibitory effects in meningiomas both in vitro and in vivo. Irinotecan was much more effective against the malignant meningioma cell line than against primary meningioma cultures. Therefore, this drug may have an important therapeutic role in the treatment of atypical or malignant meningiomas and should be evaluated further for this purpose.     

Coordinate inhibition of alkaline phosphatase and type X collagen syntheses by 1,25-dihydroxyvitamin D3 in primary cultured hypertrophic chondrocytes

Summary The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (2.3×10^-12-1.4×10^-6 [M]) on alkaline phosphatase, collagen, and cell proliferation were examined in primary cultured hypertrophic chondrocytes prepared from the distal epiphyseal growth plate of the tibias of 12-day chick embryos. 1,25(OH)₂D₃ showed time- and dose-dependent inhibitory effects on the alkaline phosphatase and collagen levels. The inhibition of alkaline phosphatase activity became detectable at 2×10^-11 [M] and reached 10% of control at 10^-7 [M]. The concentration of 1,25(OH)₂D₃ giving a 50% inhibition of the enzyme level was approximately 3×10^-10 [M]. Of the two extracellular collagen pools, a cell-associated matrix pool showed a more dramatic decrease (to 10% of control) than a culture medium pool (to 50% of control) at increased 1,25(OH)₂D₃ concentrations. The degree of inhibition was different for each type of chondrocyte-specific collagen (types II, IX, X, and XI). Types II and IX were inhibited in a parallel manner to only 60–80% of control. On the other hand, types X and XI were more greatly reduced up to 10% of control, and their dose-dependent inhibitory curves were similar to that of alkaline phosphatase. On cell proliferation, 1,25(OH)₂D₃ had a biphasic effect: stimulation at 10^-10–10^-8 [M] and inhibition at higher levels. The results revealed the significant involvement of 1,25(OH)₂D₃ in the metabolism of two probable calcification-related products, alkaline phosphatase and type X collagen.     

Hypoxia-mediated impaired differentiation by LLC-PK1 cells: evidence based on the protein kinase C profile.

We recently reported that mild hypoxia in LLC-PK1 cells, grown in standard fashion under a still layer of overlying medium at 5% CO2/18% O2 environment, result in decreased oxidative metabolism and impaired differentiated functions in comparison to adequately oxygenated cultures maintained either under a higher oxygen (36% O2) environment or conditions of continuous rocking of the media fluid. In the present study, subcellular distribution of a regulatory enzyme protein kinase C (PKC) was examined between hypoxic still and normoxic rocked LLC-PK1 cells. Subconfluent cultures of hypoxic LLC-PK1 cells exhibited significantly lower and predominantly membrane-bound PKC activity in comparison to mostly cytosolic localization of this enzyme in normoxic rocked cells. One hour of exposure of adequately oxygenated-rocked LLC-PK1 cells with the phorbol ester TPA, a dedifferentiating agent that did not effect the cell ATP content, resulted in significant inhibition of dome formation and sodium-dependent glucose transport activity, a partial loss of pH-responsive ammoniagenesis, and almost complete translocation of protein kinase C activity from cytosol to the membrane pool; all of which resembles the behavior of hypoxic still cultured cells. In addition, acute re-oxygenation of hypoxic still cultures by rocking the media fluid for one hour resulted in an increase in cell ATP content to the cellular levels of ATP observed in normoxic rocked cells. However, all the parameters of differentiation were unaffected by re-oxygenation. These studies support the notion that hypoxia can act in some primary fashion, independent of its effects on energy metabolism, to impair cellular differentiation in LLC-PK1 cells. They also raise the possibility that activation of protein kinase C may act as an important mediator in this process.