The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to <em>20</em> ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several <em>growth</em> <em>factors</em> was also tested. Insulin-like <em>growth</em> <em>factor</em> I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal <em>growth</em> <em>factor</em> inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas <em>fibroblast</em> <em>growth</em> <em>factor</em> had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Glial cell line-derived neurotrophic <em>factor</em> (GDNF), a distant member of the TGF-beta superfamily, is a survival <em>factor</em> for various neurons, making it a potential therapeutic agent for neurodegenerative disorders. Here we present the genomic structure and characterization of the promoter of the human GDNF (hGDNF) gene. It contains three exons coding for a cDNA of 4.6 kb including large 5'- and 3'-untranslated regions (UTRs). The 3'-UTR contains a polymorphic AGG repeat that appears not to be expanded in patients suffering from different neurodegenerative disorders. RT-PCR results in at least three different hGDNF transcripts including one that lacks exon 2. Transient expression experiments reveal that exon 2 is essential for proper cellular processing to yield a secreted form of hGDNF, whereas expression of exon 3 alone is sufficient to code for a mature form of hGDNF retained within the cell. Our data show that the hGDNF gene is driven by a TATA-containing promoter preceding exon 1. A second promoter element has been mapped to a region 5' of exon 2. Both promoters are in close proximity to CpG islands covering exons 1 and 2. Using luciferase as a reporter gene, the TATA-containing hGDNF promoter facilitates a <em>20</em>- to 40-fold increase in transcription when compared with a corresponding promoterless construct, whereas the second promoter confers only weak activity. Furthermore, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2, tetradecanoyl 12-phorbol acetate, an inflammatory agent, and cAMP increase promoter activity, suggesting that GDNF transcriptional regulation is a target of exogenous signals.

BACKGROUND

The leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins constitute an integral membrane protein family that has three members: LRIG1, LRIG2, and LRIG3. LRIG1 negatively regulates growth factor signaling, but little is known regarding the functions of LRIG2 and LRIG3. In oligodendroglial brain tumors, high expression of LRIG2 correlates with poor patient survival. Lrig1 and Lrig3 knockout mice are viable, but there have been no reports on Lrig2-deficient mice to date.

RESULTS

Lrig2-deficient mice were generated by the ablation of Lrig2 exon 12 (Lrig2E12). The Lrig2E12-/- mice showed a transiently reduced growth rate and an increased spontaneous mortality rate; 20-25% of these mice died before 130 days of age, with the majority of the deaths occurring before 50 days. Ntv-a transgenic mice with different Lrig2 genotypes were transduced by intracranial injection with platelet-derived growth factor (PDGF) B-encoding replication-competent avian retrovirus (RCAS)-producing DF-1 cells. All injected Lrig2E12+/+ mice developed Lrig2 expressing oligodendroglial brain tumors of lower grade (82%) or glioblastoma-like tumors of higher grade (18%). Lrig2E12-/- mice, in contrast, only developed lower grade tumors (77%) or had no detectable tumors (23%). Lrig2E12-/- mouse embryonic fibroblasts (MEF) showed altered induction-kinetics of immediate-early genes Fos and Egr2 in response to PDGF-BB stimulation. However, Lrig2E12-/- MEFs showed no changes in Pdgfrα or Pdgfrβ levels or in levels of PDGF-BB-induced phosphorylation of Pdgfrα, Pdgfrβ, Akt, or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Overexpression of LRIG1, but not of LRIG2, downregulated PDGFRα levels in HEK-293T cells.

CONCLUSIONS

The phenotype of Lrig2E12-/- mice showed that Lrig2 was a promoter of PDGFB-induced glioma, and Lrig2 appeared to have important molecular and developmental functions that were distinct from those of Lrig1 and Lrig3.

BACKGROUND

Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model.

METHODS

Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry.

RESULTS

When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates.

CONCLUSIONS

The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.

Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 <em>fibroblasts</em> overexpressing the epidermal <em>growth</em> <em>factor</em> (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell <em>growth</em>. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and <em>growth</em> of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, <em>growth</em> stimulated by EGF was inhibited, moderately (-<em>20</em>%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.

Treatment of quiescent BALB/c mouse 3T3 cells with murine interferon alpha/beta (IFN-alpha/beta) (1000 units/ml) leads to the appearance at 4 hr of 1.7-kilobase 2',5'-oligoadenylate (2',5'-OAS)mRNA as detected by Northern blot analysis. This mRNA accumulates for at least 18 hr. Two protein kinase C activators, 1,2-dioctanoyl glycerol and phorbol 12-myristate 13-acetate, suppress, whereas the calcium ionophore ionomycin enhances, the IFN-alpha/beta-induced expression of 2',5'-OAS mRNA. The 8-bromo and dibutyryl analogs of cAMP and the adenylate cyclase activator forskolin did not affect the induction of 2',5'-OAS mRNA by IFN-alpha/beta. In the absence of IFN-alpha/beta, the above agents used either singly or in combinations, did not induce 2',5'-OAS mRNA expression nor did platelet-derived <em>growth</em> <em>factor</em> (1-2 units/ml), <em>fibroblast</em> <em>growth</em> <em>factor</em> (6-100 ng/ml), or bovine serum (10-<em>20</em>%). Bovine serum also did not affect 2',5'-OAS mRNA induction by IFN-alpha/beta. The poly(ADP)-ribose synthetase inhibitor 3-aminobenzamide suppressed IFN-alpha/beta-induced 2',5'-OAS gene expression. These results suggest that in quiescent BALB/c 3T3 cells (i) the 2',5'-OAS gene is not responsive to the three major signal transduction pathways activated by diacylglycerol, Ca2+, and cAMP; (ii) induction of the 2',5'-OAS gene by IFN-alpha/beta is decreased by activation of the protein kinase C pathway but enhanced by elevation of intracellular [Ca2+].

Previously we have reported changes in <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) in conditioned medium (CM) derived from rat mammary tumours undergoing remission. We have used a similar approach to assay for the presence of FGFs in human breast tissue and cell lines. The majority of cancer tissues (35/50), benign tissues (8/9) and all cancer adjacent normal tissues (<em>20</em>/<em>20</em>) released heat labile, NR6 transforming activity which coeluted from heparin with acidic FGF (aFGF) at 0.9-1.1 M NaCl and was neutralised by antibodies to aFGF. The conclusion that the majority of breast cancers contain active aFGF was supported by immunoblotting. The CM of a minority (15/50) of cancers and one benign tissue had highly transforming activity for NR6 cells, and was mitogenic for a breast cancer cell line, was heat labile, and strongly heparin binding, eluting at 1.5-2.0 M salt. It was not immunoreactive with antibodies to aFGF, basic FGF (bFGF) or Kaposi's FGF (kFGF) and its activity was reduced by the presence of aFGF, suggesting competition for the same receptor. Very little aFGF was observed in the CM of these tumours, and neither aFGF nor other FGF activity was detected in CM of breast cell lines.

Granulation-tissue myo<em>fibroblasts</em> are important in wound contraction, disappearing in normal scars but persisting in hypertrophic scars. While transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) is implicated in excessive scarring and induces the accumulation of myo<em>fibroblasts</em>, the role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) on scarring remains unreported. Four linear full-thickness incisions, each <em>20</em> mm long, were made dorsally on 25 adult male Hooded Lister rats. In each animal, one wound was unmanipulated (control), one was injected with TGF-beta 1 (positive control), one with vehicle alone and one with FGF-2 (test). The wounds were injected daily for 5 days. Animals were sacrificed in groups of five on days 5, 10, 15, <em>20</em> and 30 after wounding. Wounds were subjected to tensiometry. Sections were stained for collagen fibres, immunostained for myo<em>fibroblasts</em> and studied under light microscopy and electron microscopy. Myo<em>fibroblasts</em> were present in the granulation tissue on day 5, reached their maximum number on day 10 and disappeared from all wounds by day 30. Treatment with FGF-2 inhibited this transient phenotypic change of granulation-tissue <em>fibroblasts</em> into myo<em>fibroblasts</em>, relative to controls (15.23% versus 58.71%, 15.23% versus 54.71% and 15.23% versus 53.15%; P<0.003 on day 10, paired t-test). Test collagen-fibre orientation resembled that of the normal dermis, in contrast to that of the control wounds. Test wound breaking strength was unreduced. These results suggest a possible anti-scarring effect of FGF-2 during wound healing.

In an effort to define the roles of bone morphogenic proteins (BMPs) and <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) during chick limb development more closely, we have implanted beads impregnated with these <em>growth</em> <em>factors</em> into chick limb buds between stages <em>20</em> and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (<em>20</em>-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. We argue that the apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these <em>growth</em> <em>factors</em> have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two <em>growth</em> <em>factor</em> families could control the size of appendages when they are initially formed.

Recombinant <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>-4 (FGF4) and FGF2 induce extracellular signal-regulated kinase-1/2 activation and DNA synthesis in murine aortic endothelial (MAE) cells. These cells co-express the IIIc/Ig-3 loops and the novel glycosaminoglycan-modified IIIc/Ig-2 loops isoforms of FGF receptor-2 (FGFR2). The affinity of FGF4/FGFR2 interaction is <em>20</em>-30 times lower than that of FGF2 and is enhanced by heparin. Overexpression of FGF2 or FGF4 cDNA in MAE cells results in a transformed phenotype and increased proliferative capacity, more evident for FGF2 than FGF4 transfectants. Both transfectants induce angiogenesis when applied on the top of the chick embryo chorioallantoic membrane. However, in contrast with FGF2-transfected cells, FGF4 transfectants show a limited capacity to <em>growth</em> under anchorage-independent conditions and lack the ability to invade 3D fibrin gel and to undergo morphogenesis in vitro. Also, they fail to induce hemangiomas when injected into the allantoic sac of the chick embryo. In conclusion, although exogenous FGF2 and FGF4 exert a similar response in MAE cells, significant differences are observed in the biological behavior of FGF4 versus FGF2 transfectants, indicating that the expression of the various members of the FGF family can differently affect the behavior of endothelial cells and, possibly, of other cell types, including tumor cells.

Cell lines were successfully established in continuous suspension culture from 10 patients with a histopathological diagnosis of diffuse histiocytic lymphoma (SU-DHL-1 to SU-DHL-10), two with North American Burkitt's lymphoma (SU-AmB-1 and SU-AmB-2), and one with acute lymphoblastic leukemia (SU-ALL-1). By screening a variety of parameters, including media, sera, effusion fluids, feeder layers, and chemical supplements, the nutritive <em>growth</em> requirements of lymphoma cells obtained from malignant effusions and lymph node biopsies were determined for each tumor. Most of these cell lines initially required human skin <em>fibroblast</em> or epithelial cell feeder layers from which they could be weaned after one to six weeks in culture and maintained in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing <em>20</em>% fetal calf serum and 10% pooled human serum. Several of these cell lines were successfully cloned on 0.5% Noble agar substrates. In the presence of human serum and selected feeder monolayers, cloning efficiencies increased significantly from less than 1% to 15 to 25%. In addition, the cloning efficiencies of certain cell lines showed a concentration-dependent increase with specific chemical supplements including L-cysteine and dithiothreitol. Placental colony-stimulating <em>factor</em>, nerve <em>growth</em> <em>factor</em>, epithelial <em>growth</em> <em>factor</em>, and <em>fibroblast</em>ic <em>growth</em> <em>factor</em> were ineffective in augmenting the cloning efficiencies of the human lymphoma cell lines. After a single passage on agar, cells subpassaged from visible colonies showed markedly increased cloning efficiencies to levels as high as 50%. Such cloning efficiencies, coupled with the use of replica plating, make this technique applicable to genetic and quantitative radiobiological, immunological, and chemotherapeutic studies. Although these methods have thus far been used only with lymphoreticular tumors, they may also be applicable to the cell culture of other human neoplasms and normal tissues.

Anti-angiogenic therapy mediated by drugs and food components is an established strategy for cancer prevention. Our previous cell-culture studies identified a food-derived anti-angiogenic compound, tocotrienol (T3, an unsaturated vitamin E), as a potential angiogenic inhibitor. Among T3 isomers, delta-T3 is considered as the most potent compound. The purpose of this study was therefore to evaluate the inhibitory effect of delta-T3 on tumor angiogenesis. As <em>growth</em> <em>factors</em> (e.g., vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>) play critical roles in tumor angiogenesis, a conditioned medium rich in these <em>growth</em> <em>factors</em> from human colorectal adenocarcinoma cells (DLD-1-CM) was used as an angiogenic stimulus. Delta-T3 (2.5-5 microM) significantly suppressed DLD-1-CM-induced tube formation, migration, and adhesion on human umbilical vein endothelial cells. These effects were partly associated with reactive oxygen species generation by delta-T3. Western blot analysis revealed that the anti-angiogenic effect of delta-T3 is attributable to regulation of <em>growth</em> <em>factor</em>-dependent phosphatidylinositol-3 kinase (PI3K)/phosphoinositide-dependent protein kinase (PDK)/Akt signaling as well as to induction stress response in endothelial cells. Moreover, we conducted an in vivo mouse Matrigel plug angiogenesis assay, and found that delta-T3 (10-<em>20</em> microg) exhibits dose-dependent inhibition of DLD-1-induced vessel formation. These results suggest that T3 has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.

We tested the hypothesis that atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (AII) could be prevented by the induction of heat-shock protein 72 (HSP72) by hyperthermia (HT). In cultured atrial <em>fibroblasts</em> isolated from male Sprague-Dawley rats, HT (42 degrees C) was applied for 30 min. AII (100 nmol/L) was added to the medium 8 h later. HT induced the expression of HSP72, which was associated with the attenuation of AII-induced extracellular signal-regulated kinase (ERK1/ERK2) phosphorylation, alpha-smooth muscle actin (alpha-SMA) expression, transforming <em>growth</em> <em>factor</em>-beta(1) secretion, collagen synthesis, and expression of collagen type I and tissue inhibitor of metalloproteinases-1. A small interfering RNA targeting HSP72 abolished these anti-fibrotic effects of HT. In male Sprague-Dawley rats in vivo, an osmotic mini-pump was subcutaneously implanted for continuous infusion of AII (400 ng/kg/min). Whole-body HT (43 degrees C, <em>20</em> min) was applied 24 h before and 7, 14, and 21 days after the start of the AII infusion. Repeated HT led to the induction of HSP72 expression, which resulted in an attenuation of AII-induced left atrial fibrosis. In an electrophysiological study using isolated perfused heart, continuous AII caused slowing of interatrial conduction without affecting atrial refractoriness. In AII-treated hearts, extrastimuli from the right atrial appendage resulted in a high incidence of repetitive atrial responses, which were suppressed by treatment with HT. Our results suggest that HT treatment is effective in suppressing AII-mediated atrial fibrosis and AF via induction of HSP72 at least in parts, and is thus expected to be a novel strategy for prevention of AF.

Renal cell carcinoma (RCC) is an angiogenic tumor resistant to standard cytotoxic chemotherapeutic agents. Although often responsive to immunomodulatory agents including interleukin 2 and IFN-alpha, the overall results in randomized Phase III studies are disappointing with only modest improvements in overall survival. This Phase II study evaluated the efficacy and tolerability of razoxane, an antiangiogenic topoisomerase II inhibitor, in 40 patients (32 men, 8 women; age: range, 31-76 years; median, 58 years) with inoperable RCC. Twenty patients received razoxane 125 mg p.o., twice a day for 5 days each week for 8 weeks (one cycle). This was repeated in patients with stable disease (StD), but was discontinued after 16 weeks if there was no evidence of an objective response. Because minimal toxicity was seen, subsequent patients (n = <em>20</em>) were treated until progressive disease (PD) was documented. Of 38 evaluable patients, 11 (29%) had StD for a minimum of 4 months, and the remainder had PD. Median overall survival was 7.3 months. Duration of survival was significantly better in patients with StD compared with those with PD (P = 0.003). The effect of treatment on six potential surrogate serum/plasma (vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), urokinase plasminogen activator soluble receptor (uPAsr), E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand's <em>factor</em> (vWF) and two urinary (VEGF and bFGF) markers of angiogenesis was evaluated before and after 1 cycle of treatment. Pretreatment serum VEGF and E-selectin levels above the median value were associated with a poor prognosis. Serum VCAM-1 levels and urinary VEGF levels rose significantly after one cycle in patients with PD but not in those with StD. Serum VEGF, bFGF, VCAM-1 and vWF, plasma uPAsr and urinary bFGF levels were significantly higher in PD patients compared with StD patients before and/or after 1 cycle of treatment. In conclusion, razoxane is an antiangiogenic agent that has minimal toxicity and that requires further evaluation in combination with other active agents in the treatment of RCC. Surrogate serum and urinary markers of angiogenesis may have a role to play in predicting disease response and overall survival in RCC.

A method for establishing primary cultures of smooth muscle cells (SMCs) from the porcine coronary artery without either microdissection and/or enzymatic dispersion was developed using selective migration of cells from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and adherence ability to separate SMC from contaminating <em>fibroblasts</em> or endothelial cells. The cell type was determined by immunohistochemical staining with monoclonal antibodies to SM alpha-actin, SM myosin, h-caldesmon and von Willebrand <em>factor</em>. The first wave of migration (1-7 days) consisted of a mixture of <em>fibroblasts</em> and SMCs. Only SMCs were present in the second wave of migration (7-14 days). Endothelial cells, which exhibited a lower capacity for migration and adherence, were restricted to the third wave of migration (14-21 days). Cells obtained from the second wave of migration exhibited the characteristic single-layered, aligned, hill-and-valley pattern of SMCs when confluent. Quiescence was attained 4-5 days after removal of serum, as established by [3H]-thymidine incorporation. Stimulation of the quiescent SMCs with <em>20</em>% FBS resulted in a synchronous re-entry into the cell-cycle with S phase reached 15-18 h later. The SMCs prepared using this protocol thus exhibit the structural markers and capacity to undergo phenotypic modulation that are characteristic of SMCs in vivo. This approach to establishing primary cultures of SMCs offers the advantage of selecting for the subpopulation of cells capable of migration in response to injury or <em>growth</em> <em>factor</em> stimulation.

OBJECTIVE

To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas.

METHODS

Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways.

RESULTS

Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands.

CONCLUSIONS

The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

OBJECTIVE

Blocking intestinal bile acid (BA) absorption by inhibiting or inactivating the apical sodium-dependent BA transporter (Asbt) classically induces hepatic BA synthesis. In contrast, blocking intestinal BA absorption by inactivating the basolateral BA transporter, organic solute transporter alpha-beta (Ostα-Ostβ) is associated with an altered homeostatic response and decreased hepatic BA synthesis. The aim of this study was to determine the mechanisms underlying this phenotype, including the role of the farnesoid X receptor (FXR) and fibroblast growth factor 15 (FGF15).

METHODS

BA and cholesterol metabolism, intestinal phenotype, expression of genes important for BA metabolism, and intestinal FGF15 expression were examined in wild type, Ostα(-/-), Fxr(-/-), and Ostα(-/-)Fxr(-/-) mice.

RESULTS

Inactivation of Ostα was associated with decreases in hepatic cholesterol 7α-hydroxylase (Cyp7a1) expression, BA pool size, and intestinal cholesterol absorption. Ostα(-/-) mice exhibited significant small intestinal changes, including altered ileal villus morphology, and increases in intestinal length and mass. Total ileal FGF15 expression was elevated almost 20-fold in Ostα(-/-) mice as a result of increased villus epithelial cell number and ileocyte FGF15 protein expression. Ostα(-/-)Fxr(-/-) mice exhibited decreased ileal FGF15 expression, restoration of intestinal cholesterol absorption, and increases in hepatic Cyp7a1 expression, fecal BA excretion, and BA pool size. FXR deficiency did not reverse the intestinal morphological changes or compensatory decrease for ileal Asbt expression in Ostα(-/-) mice.

CONCLUSIONS

These results indicate that signaling via FXR is required for the paradoxical repression of hepatic BA synthesis but not the complex intestinal adaptive changes in Ostα(-/-) mice.

The MMP, matrilysin (MMP-7), has been shown to be overexpressed in prostate cancer cells and to increase prostate cancer cell invasion. Prostate stromal <em>fibroblasts</em> secrete <em>factor</em>(s), including <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) that induces promatrilysin expression in LNCaP cells. In the present study, we investigated the signal transduction pathway involved in the FGF-1-induced expression of promatrilysin. FGF-1 treatment significantly increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). This induction was time-dependent and was sustained until 24 hours after treatment. Treating the cells with MEK1/2 inhibitor (PD98059) eliminated ERK activation completely and blocked FGF-1-mediated induction of promatrilysin expression. Transient transfection studies with human matrilysin promoter resulted in a four- to five-fold increase in reporter luciferase enzyme activity that was blocked by the MEK1/2 inhibitor (PD98059). Serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was observed after FGF-1 treatment and pretreatment with <em>20</em> microM PD98059-abolished STAT3 phosphorylation. Transient transfection with dominant negative STAT3 inhibited FGF-1-induced transactivation of the matrilysin promoter indicating that STAT3 plays an important role in FGF1-induced matrilysin expression. We propose that the FGF-1-induced signaling pathway that leads to promatrilysin expression is ERK-dependent and leads to phosphorylation of Ser-727 on STAT3, phosphorylated STAT3, then binds and transactivates the matrilysin promoter. Our results demonstrate that ERK-MAP kinase and transcription <em>factor</em> STAT3 are important components of FGF-1-mediated signaling, which induce promatrilysin expression in LNCaP cells.

Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are an important source for various cytokines and <em>growth</em> <em>factors</em>. This study was conducted to analyze the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and release basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and to characterize its regulation by inflammatory cytokines. HPMCs constitutively synthesized and released considerable amounts of bFGF as detected by a specific immunoassay. Almost 80% of bFGF (1547 +/- 173 pg/10(5) cells) was localized intracellularly. Approximately <em>20</em>% of the bFGF (357 +/- 27 pg/10(5) cells) was associated with extracellular matrix components on the HPMC surface. Small amounts of bFGF (<1%) were detectable in tissue culture supernatants (8.4 +/- 1.4 pg/10(5) cells). Treatment of HPMCs with interleukin-1beta (IL-1beta; 1 ng/ml) resulted in a significant increase in bFGF production. The intracellular bFGF content showed a rapid but only transient increase, which was significant above background levels after 24 hours (41% increase; P < 0.05). This increase in intracellular bFGF concentration was associated with an induction of the release of bFGF. Within 96 hours, the release of bFGF to the cell surface and into the supernatant increased by 58% (564 +/- 52.4 pg/10(5) cells; P < 0.01) and by 214% (26.4 +/- 3.2 pg/10(5) cells; P < 0.001), respectively. Neither tumor necrosis <em>factor</em>-alpha nor interferon-gamma affected bFGF synthesis by HPMCs. Stimulation of HPMCs with IL-1beta increased steady-state levels of bFGF-specific mRNA. Immunohistochemical analyses of peritoneal tissue revealed constitutive expression of bFGF by HPMCs. This in situ expression proved to be most pronounced in areas of serosal inflammation in activated HPMCs. Our study demonstrates that HPMCs synthesize and release significant amounts of bFGF and that the expression of this <em>growth</em> <em>factor</em> is significantly up-regulated by the proinflammatory cytokine IL-1beta. The data support the view that HPMCs are key regulators of abdominal disease processes such as peritonitis, peritoneal fibrosis, or peritoneal tumor metastasis.

The purpose of this study was to determine the subpopulation dynamics of human colon carcinoma (HCC) cells <em>growing</em> at orthotopic (cecum, liver) or ectopic (subcutis, kidney, spleen) sites in nude mice and to correlate any out<em>growth</em> of distinct clones with the differential expression of metastasis-related genes. Low metastatic KM12C HCC cells were genetically tagged with a retrovirus harboring the neomycin-resistance (Neo(R)) gene. Southern blot analyses demonstrated only minor resolution of the Neo(R) hybridization pattern in DNA isolated from primary tumors <em>growing</em> orthotopically or ectopically, suggesting a polyclonal out<em>growth</em>. In contrast, a major resolution of the Neo(R) hybridization pattern was observed in liver-specific metastases, demonstrating the out<em>growth</em> of single dominant clones. Expression of epidermal <em>growth</em> <em>factor</em> receptor (EGR-R) increased <em>20</em>-60% in the liver metastases vs spleen tumors and the KM12C Neo(R) cells. Transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha), amphiregulin (AR), and c-met showed only modest differences in mRNA expression. A <em>20</em>-80% increase in type IV collagenase mRNA levels was also observed in all tumor specimens. Furthermore, expression of the multi-drug resistance gene PGY-1 and the carcinoembryonic antigen (CEA) gene were elevated in the liver metastases compared with the spleen tumors and cultured cells. Transcript levels of the angiogenic <em>factors</em> interleukin-8 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> did not correlate with clonal out<em>growth</em>. These data demonstrate a correlation between EGF-R, type IV collagenase, CEA, and PGY-1 gene expression and the production of liver metastases. Our results suggest that distinct HCC clones differentially expressing specific mRNA transcripts for metastasis-related genes are the forerunners of the experimental liver metastatic lesions.

Melanocytes derived from fetal or adult skin do not propagate in vitro unless cultured in the presence of <em>factors</em> such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In a search for physiological <em>factors</em> regulating the <em>growth</em> of melanocytes, extracts of various cultured cell types were tested. <em>Factors</em> produced by melanoma and astrocytoma cell lines support continued proliferation of melanocytes in the absence of TPA. WI-38, a <em>fibroblast</em> cell line derived from human embryonic lung, was the most active source of melanocyte <em>growth</em> <em>factors</em>. No melanocyte <em>growth</em>-promoting activity was found in extracts of cultured neuroblastoma, renal cancer, normal keratinocytes, or renal epithelium. Nerve <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, melanocyte-stimulating hormone, transforming <em>growth</em> <em>factor</em>-beta, and platelet-derived <em>growth</em> <em>factor</em> did not have <em>growth</em>-promoting activity for melanocytes. The presence of melanocyte <em>growth</em> <em>factors</em> and TPA together resulted in the strongest mitogenic activity for melanocytes, permitting the recovery (at <em>20</em> days) of 4 to <em>20</em> times as many cells as in <em>growth</em> <em>factor</em> or TPA alone.

A low metastatic clone, G6, was isolated from the B16 melanoma cell line by cloning procedure. When the cells were cultured in vitro with <em>fibroblasts</em> from newborn mice, the lung-colonizing potential of G6 cells was substantially increased. The effect of coculture depended on the number of the <em>fibroblasts</em>. The elevated colonizing potential of G6 cells was reversed to the original low potential by subculturing them for <em>20</em> days without the <em>fibroblasts</em>. The culture medium conditioned by G6-<em>fibroblast</em> coculture demonstrated an activity to enhance the lung-colonizing potential of G6 cells, whereas the medium from the culture of <em>fibroblasts</em> alone showed only a little activity. The <em>growth</em> rate and plating efficiency of G6 cells cultured with the <em>fibroblasts</em> or in the conditioned medium did not differ from those of uncocultured G6 cells. The potentiating activity in the conditioned medium was nondialyzable and stable to heating at 80 degrees C for 10 min, but was lost after heating for 10 min at 1<em>20</em> degrees C, or by the treatment with trypsin. These results indicate that the enhancement of lung-colonizing potential of G6 cells could be mediated by a soluble <em>factor</em>(s) released from cocultured <em>fibroblasts</em>.

Liver fibrosis plays a pivotal role in liver function impairment and is a feature of chronic infection by hepatitis C virus (HCV). Ten to <em>20</em>% of patients progress to cirrhosis leading to an increased risk of liver failure or hepatocellular carcinoma (HCC). Recent advances have culminated in clear evidence that fibrosis can be reversible, and in expectation that effective anti-fibrotic therapy will improve the prognosis. Among the different cellular pathways involved in fibrosis, the transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) signaling plays a major role. Increases of TGF-beta1 expression is associated with fibrotic diseases and this cytokine could be a target for an antifibrotic drug. Clinical results on radiation-induced fibrosis have brought some evidences that Cu-Zn SOD (SOD1) could be an anti-fibrotic drug. Its therapeutic effect could be related to a down-regulation of TGF-beta1 as proved in a well characterized pig model of radiation induced fibrosis, where the efficacy of Cu-Zn SOD has been shown in reversing fibrosis. Using 3-D skin co-culture of <em>fibroblasts</em> from this pig model, it was showed that SOD significantly reduces the expression of the TGFbeta1, both at the mRNA and protein level. An experimental work could be undertaken to validate the Cu-Zn SOD as an anti-fibrotic drug, using HCV core protein expressing transgenic mice. As the current anti-HCV therapy with pegylated interferon combined with ribavirin can eradicate virus and stop the progression of fibrosis, but near 50% of HCV infected patients are non responders, a controlled trial is planned for HCV infected patients non responders to this therapy with a Metavir fibrosis score reaching F = 2 or >2.

Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived <em>growth</em> <em>factor</em> (5 micrograms/ml), epidermal <em>growth</em> <em>factor</em> (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term serial culture (17.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell <em>growth</em> that varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by arachidonate and thrombin by 17 to <em>20</em> and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult endothelial cells were 2 to 3 times that of human adult smooth muscle cells and <em>20</em> to 30 times that of human <em>fibroblasts</em>.