We have determined the nucleotide sequence of the human <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) gene, including 800 bp of the 5'-flanking region and compared the sequence with the previously published murine Fgfr3 gene. The organization of the gene is highly conserved between man and mouse. We used the intron sequences to design a set of primers that allow amplification of the <em>17</em> exons (2-18) that encode the complete open reading frame. Using these primers the FGFR3 gene can be amplified at the genomic level, which significantly facilitates mutational screening.

We have previously demonstrated that tooth size is determined by dental mesenchymal <em>factors</em>. Exogenous bone morphogenetic protein (BMP)4, Noggin, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-<em>17</em>, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.

Angiogenic <em>factors</em> play a role in tumor <em>growth</em> and spread. The object of this study was to analyze the correlation between mRNA expression of angiogenesis-related genes and disease outcome in advanced-stage ovarian carcinomas. Sections from 66 primary ovarian carcinomas and metastatic lesions from 41 patients diagnosed with advanced stage ovarian carcinoma (FIGO stages III-IV) were evaluated for expression of basic <em>fibroblast</em> <em>factor</em> (bFGF), interleukin-8 (IL-8), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) using mRNA In Situ Hybridization (ISH). Patients were divided in two groups based on disease outcome. Long-term survivors (<em>17</em> patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period was 70 months. The mean values for DFS and OS were 116 and 133 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Expression of bFGF mRNA, most often intense, was detected in tumor and stromal cells in the majority of cases. Weak expression of IL-8 mRNA was detected in both cell compartments, while VEGF mRNA expression was limited to few cases. Primary tumors displayed higher bFGF and IL-8 mRNA expression. However, these differences did not reach statistical significance (P>> 0.05). bFGF, IL-8 and VEGF mRNA expression in both tumor and stromal cells was comparable in tumors of long-term and short-term survivors, and showed no correlation with disease outcome in survival analysis (P>> 0.05). bFGF is the major angiogenic <em>factor</em> expressed in ovarian carcinoma at the mRNA level. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma.

The fibrotic and antiapoptotic effects of insulin-like <em>growth</em> <em>factors</em> (IGF) are mediated by type I IGF receptor (IGF-1R). IGFs could play a role in intestinal stricturing and in the maintenance of inflammation in Crohn's disease (CD). We aimed to describe IGF-1R expression in CD intestinal lesions, to compare it to other intestinal inflammatory diseases and to correlate it with fibrosis and apoptosis. IGF-1R expression and apoptosis (active caspase-3) were studied by immunohistochemistry. Surgical intestinal specimens [<em>17</em> CD, nine controls, six diverticulitis and four ulcerative colitis (UC)] were used. IGF-1R was expressed transmurally mainly by inflammatory cells (IC) and smooth muscle cells, both in diseased intestine and controls. IGF-1R positive IC were increased in the mucosa and the submucosa of CD (P < 0.007), and in involved areas compared to uninvolved areas (P = 0.03). In UC, the number of IGF-1R positive IC was only increased in the mucosa, and was not different from controls in the submucosa. In diverticulitis, the number of IGF-1R positive IC did not differ from controls. In CD submucosa, IGF-1R expression in IC was inversely correlated with apoptosis in uninvolved areas (P = 0.01). Expression of IGF-1R in submucosal <em>fibroblast</em>-like cells, subserosal adipocytes and hypertrophic nervous plexi was specific for CD. We have shown a transmural altered expression of IGF-1R in CD. This may suggest a role for IGF-1R in the maintenance of chronic inflammation and stricture formation in CD.

<em>Growth</em> of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1:1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 micrograms/ml glutathione, 10 micrograms/ml insulin, 10 micrograms/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 micrograms/ml ethanolamine, 20 ng/ml epidermal <em>growth</em> <em>factor</em>, 2.0 nM <em>17</em> beta-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 micrograms/ml Tf, and 200 micrograms/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like <em>growth</em> <em>factor</em> I (IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these <em>factors</em> in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At less than or equal to ng/ml concentrations, epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> II, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of <em>growth</em>-promoting <em>factors</em> on human breast cancer cells without interfering activities known to be present in serum.

The establishment of a propagating cell culture system of adipocyte precursors is facilitating the elucidation of the regulation of fat cell development by hormones and <em>growth</em> <em>factors</em>. <em>17</em> beta-Estradiol significantly enhances the replication of cultured human omental adipocyte precursors. Androgens do not influence their <em>growth</em>. These findings suggest that estrogens account, at least partly, for the changes that occur in certain adipose tissue regions at puberty. While the physiological role of <em>fibroblast</em> <em>growth</em> <em>factor</em> is unknown, the bovine polypeptide increases the replicative rate of skin <em>fibroblasts</em> to a greater extent than that of adipocyte precursors. On the other hand, partially purified, novel bovine pituitary polypeptides promote the replication of human adipocyte precursors to an appreciably greater extent than <em>fibroblast</em> <em>growth</em> <em>factor</em>. We are further characterizing these putative 'adipocyte <em>growth</em> <em>factors</em>'. Porcine pancreatic polypeptide significantly inhibits the multiplication of human omental adipocyte precursors. These findings may account for the scarcity of pancreatic fat tissue, and suggest that pancreatic polypeptide may control the <em>growth</em> of adipose tissue through its inhibitory effect.

A soluble form of the insulin-like <em>growth</em> <em>factor</em> II/mannose 6-phosphate receptor (sIGF-II/MPR) is present in fetal bovine serum and carries mature 7.5-kDa insulin-like <em>growth</em> <em>factor</em> II (IGF-II) and at least 12 different high molecular weight (Mr) IGF-II isoforms (Valenzano, K. J., Remmler, J., and Lobel, P. (1995) J. Biol. Chem. 270, 16441-16448). In this study, we used gel filtration and anion exchange chromatographies to resolve the isoforms into eight fractions that were characterized with respect to their biochemical, biophysical, and biological properties. Each fraction contained one to three major protein species with apparent sizes ranging from 11 to <em>17</em> kDa by SDS-polyacrylamide gel electrophoresis. The 11-kDa species contains no post-translational modifications and consists of an extended IGF-II backbone terminating at Gly-87. The remaining high Mr IGF-II isoforms are also composed of an 87-amino acid IGF-II peptide backbone but contain increasing amounts of sialated, O-linked sugars. Plasmon resonance spectroscopy experiments revealed that all the high Mr isoforms and mature 7.5-kDa IGF-II bound to immobilized recombinant soluble human IGF-I receptor, recombinant human IGF-binding protein 1, and sIGF-II/MPR with similar kinetics. In addition, radiolabeled tracer experiments demonstrated that both mature and high Mr IGF-II isoforms have similar binding profiles in fetal bovine serum and have similar affinities for IGF-II-binding proteins secreted from human <em>fibroblasts</em>. Finally, the biological activity of high Mr IGF-II was shown to be similar to or slightly better than mature IGF-II in stimulating amino acid uptake in <em>fibroblasts</em> and in inducing myoblast differentiation.

The circulating levels of several angiogenic cytokines [angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), angiogenin and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)] were evaluated in <em>17</em>4 consecutive patients with newly diagnosed, symptomatic, multiple myeloma (MM). Circulating levels of Ang-1/Ang-2 were reduced in myeloma patients compared to controls, whereas VEGF and angiogenin levels were increased. Reduced angiopoietin-1/angiopoietin-2 ratio correlated with advanced disease features including international staging system (ISS)-3 stage, renal impairment and extensive bone disease. Based on immunohistochemical results in 20 patients (10 with the higher and 10 with the lower values of circulating angiopoietin-2) we found that angiopoietin-2 is expressed by myeloma cells and correlates with increased microvessel density in subsets of patients. Furthermore, Ang-1/Ang-2 ratio correlated with survival. Patients with circulating Ang-1/Ang-2 below or equal to the median value (6.03) had a median survival of 26.3 months compared to 53 months of all others (p = 0.002). Interestingly, this was mainly observed in patients who received first-line therapy with novel agent-based regimens (65% of our patients). Furthermore, a subset of ISS-3 patients with serum Ang-1/Ang-2 above the median value had favourable prognosis (median survival: 45 months versus <em>17</em> months of all others; p = 0.0001). The multivariate analysis revealed that low Ang-1/Ang-2 ratio could independently predict for inferior survival in our cohort of patients (relative risk (RR) 2.07, 95% CI 1.50-2.42; p < 0.001). These results highlight the role of angiopoietins pathway in the biology of MM and reveal novel targets for the development of antimyeloma agents.

We identified rat developing arteries and neural crest derivatives with multiple epidermal <em>growth</em> <em>factor</em>-like domains (DANCE) as a developmentally regulated gene using suppression-subtractive hybridization. Northern analysis confirmed a fivefold induction of this mRNA transcript between fetal day 18 and 20 that persisted through postnatal day <em>17</em>. The level was declining at postnatal day 21 and was similar in adult lung to that at fetal day 18. In adults DANCE mRNA abundance was highest in lung, kidney, and spleen, lower in heart, skeletal muscle, and brain, but absent from liver and thymus. It was abundant in pulmonary artery endothelium and a lung epithelial type 2 cell line, barely detectable in vascular smooth muscle, and absent in <em>fibroblasts</em>. In situ hybridization revealed a regulated pattern of expression in endothelial cells of fetal, postnatal, and adult lung. Because DANCE mRNA was inducible in systemic arteries during recovery from injury, we searched for induction in lung injured by hyperoxia. Mouse DANCE mRNA abundance was unchanged during an acute 3-day exposure period, induced threefold 5 days into the recovery phase, and returned to baseline at days 8, 11, and 14. In situ hybridization at day 5 suggested a diffuse pattern of induction. DANCE may play a role in lung endothelial cell biology during development repair after injury.

In order to further understand the role(s) of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) in the development, differentiation and function of the central nervous system, we analysed the expression of the mRNA, and the presence and tissue distribution of the translated product, of one member of the FGF family, acidic FGF (aFGF), within the mammalian retina. Firstly, the relative abundance of aFGF mRNA was assayed in embryonic (between 14 and <em>17</em> days of gestation), postnatal (between 1 and <em>17</em> days after birth) and adult rat retina by quantitative reverse transcription-coupled polymerase chain reaction amplification using specific aFGF oligonucleotides. The level of expression remained uniformly low throughout the embryonic period and until postnatal day 7. Therefore the quantity of aFGF mRNA increased rapidly, reaching 80% of adult levels by eye opening (postnatal day 13). Adult levels were three-fold higher than at early developmental times. In situ hybridization of adult rat retina using specific antisense aFGF riboprobes revealed labelling in all cellular layers. Antisera raised against recombinant human aFGF revealed very little labelling of 4-day postnatal retina, but by postnatal days 8 and <em>17</em> immunoreactive aFGF was localized mainly within the photoreceptor cell bodies. Western blots of retinal extracts derived from <em>17</em>-day embryonic, 4-day postnatal and adult retina probed with the same antibody revealed a single immunoreactive band of the expected molecular weight (18 kDa) in all extracts. Thus aFGF is mostly transcribed and translated within the retina subsequent to the major steps of cell birth, migration and differentiation, and seems to be abundantly expressed by maturing photoreceptor cells.

It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase-polymerase chain reaction was performed at 8 and <em>17</em> days post-rupture to assess different <em>growth</em> <em>factors</em> [brain-derived neurotrophic <em>factor</em> (BDNF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), nerve <em>growth</em> <em>factor</em> (NGF) and insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia-inducible <em>factor</em>-1alpha (HIF-1alpha)] in the healing region. At 8 days post-injury, tendon mRNA levels were comparable in both groups. However, by day <em>17</em>, the mRNA levels for BDNF, bFGF, COX 1 and HIF-1alpha in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There were no significant differences in the expression of NGF, IGF-1 or COX 2 between the different groups, indicating that injury-associated expression of these molecules is not overtly influenced by loading. This study supports the notion that prolonged immobilization post-rupture hampers the healing process by compromising the up-regulation of repair gene expression in the healing tendon. It might be speculated that a shorter period of immobilization, i.e. 1 week, would not impair the healing process significantly. The findings support the current development of earlier and more active rehabilitation programs after tendon injuries.

BACKGROUND

Transforming growth factor betas (TGF-betas) are a group of homologous polypeptides that exert pleiotropic effects on various cell types and stimulate the formation of extracellular matrix and fibrosis. To evaluate whether TGF-beta isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) and their receptors (types I-III) are also of importance in the pathophysiology of liver cirrhosis, we analysed their concomitant expression and localization in human liver cirrhosis.

METHODS

Cirrhotic liver tissue samples were obtained from 17 patients (four women, 13 men) with a median age of 41 years (range 22-67). Normal liver tissues from ten patients (seven women, three men) with a median age of 55 years (range 45-75) served as controls.

METHODS

The tissues were fixed in Bouin's solution and paraffin-embedded for histological analysis. For RNA analysis, freshly obtained tissue samples were snap-frozen in liquid nitrogen and stored at -80 degrees C until analysed. Northern blot analysis was used to examine the expression of TGF-beta1, beta2 and beta3 and their receptors, type I (TbetaR-I), type II (TbetaR-II) and type III (TbetaR-III). Immunohistochemistry was performed to determine the localization of the corresponding proteins in the normal and the cirrhotic liver.

RESULTS

Northern blot analysis revealed enhanced expression (P < 0.05) of TGF-beta1 (twofold increase), TGF-beta2 (threefold increase) and TGF-beta3 (8.5-fold increase) and of TbetaR-II (threefold increase) mRNA in liver cirrhosis in comparison with normal controls. In contrast, TbetaR-I (ALK-5) and TbetaR-III mRNA expression showed no significant changes. No TGF-beta isoform immunoreactivity was present in hepatocytes in either normal livers or in liver cirrhosis. However, in liver cirrhosis, intense TGF-beta1 immunoreactivity was present in bile duct and ductular epithelial cells (including ductular proliferations) and in inflammatory cells. In a few sinusoidal lining cells, faint TGF-beta1 and moderate TGF-beta2 immunoreactivity was present. TGF-beta3 immunostaining was present in bile duct and ductular epithelial cells, in inflammatory cells and in fibroblast-like spindle cells in liver cirrhosis. For TbetaR-I and TbetaR-II, the immunoreactivity was localized in hepatocytes and biliary cells in normal and cirrhotic liver tissues, with higher intensity for TbetaR-II in the cirrhotic liver.

CONCLUSIONS

Enhanced expression of all three TGF-bea isoforms and of TbetaR-II in liver cirrhosis suggests their involvement in this fibrotic disorder. The higher immunoreactivity of the three TGF-beta isoforms in the bile duct epithelial cells in cirrhotic tissues suggests a possible role of these cells in the pathogenesis of liver cirrhosis.

A potent <em>growth</em> <em>factor</em>, PC13 embryonal carcinoma-derived <em>growth</em> <em>factor</em> (ECDGF), has been isolated from serum-free medium conditioned by PC13 murine embryonal carcinoma cells. ECDGF is a single chain, cationic hydrophobic molecule of <em>17</em> 500 daltons. ECDGF will induce DNA synthesis in established <em>fibroblast</em> cell lines and the immediate differentiated progeny of PC13 EC cells in vitro, and consequently appears to differ from other well characterised <em>growth</em> <em>factors</em> both in structure and action.

Interleukin-6 (IL-6) is a pleiotropic mediator of immune function and a <em>growth</em> <em>factor</em> for a variety of hematopoietic cell types. Because IL-1 beta is known to induce IL-6 production in nonplacental mesenchymal cells and is locally produced by maternal decidua, this study was designed to determine whether IL-1 beta could regulate IL-6 production by second trimester placental villous core mesenchymal cells (VCMC) in vitro. VCMC were prepared for culture by enzymatic digestion of placentas (14-20 weeks gestation; n = 7). Immunohistochemistry performed on the confluent cells demonstrated that more than 95% of the cells had a <em>fibroblast</em>-like morphology and were vimentin positive, less than 5% were leukocyte common antigen (CA-45) positive, and no trophoblast contamination was demonstrated by the lack of cytokeratin staining. In dose-response experiments, a specific dose-response induction of IL-6 mRNA expression and IL-6-immunoreactive protein production by IL-1 beta was demonstrated; this was first seen at 100 pg/ml IL-1 beta [455 +/- 191 ng/ml (+/- SEM); controls, 42 +/- 16 ng/ml; P < 0.05]. In time-course studies, the addition of 10 ng/ml IL-1 beta significantly increased IL-6 production rates; this was first seen at 8 h of culture and increased in a linear fashion up to 48 h. At 48 h of culture, IL-6 levels were <em>17</em> times higher in treated VCMC (861 +/- <em>17</em>9 ng/ml) compared to those in nontreated VCMC (51 +/- 14 ng/ml). In summary, IL-1 beta stimulates VCMC IL-6 production in a specific dose- and time-dependent manner. From these results, we conclude that VCMC are an important source of IL-6 in second trimester placenta and that production of placental IL-6 be may regulated by decidual IL-1 beta.

BACKGROUND

In the current study, the authors present a comprehensive genomic profile (CGP)-based study of advanced urothelial carcinoma (UC) designed to detect clinically relevant genomic alterations (CRGAs).

METHODS

DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections from 295 consecutive cases of recurrent/metastatic UC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 688X for all coding exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer, using process-matched normal control samples as a reference. CRGAs were defined as GAs linked to drugs on the market or currently under evaluation in mechanism-driven clinical trials.

RESULTS

All 295 patients assessed were classified with high-grade (International Society of Urological Pathology classification) and advanced stage (stage III/IV American Joint Committee on Cancer) disease, and 294 of 295 patients (99.7%) had at least 1 GA on CGP with a mean of 6.4 GAs per UC (61% substitutions/insertions/deletions, 37% copy number alterations, and 2% fusions). Furthermore, 275 patients (93%) had at least 1 CRGA involving 75 individual genes with a mean of 2.6 CRGAs per UC. The most common CRGAs involved cyclin-dependent kinase inhibitor 2A (CDKN2A) (34%), <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) (21%), phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (20%), and ERBB2 (<em>17</em>%). FGFR3 GAs were diverse types and included 10% fusions. ERBB2 GAs were equally divided between amplifications and substitutions. ERBB2 substitutions were predominantly within the extracellular domain and were highly enriched in patients with micropapillary UC (38% of 32 cases vs 5% of 263 nonmicropapillary UC cases; P<.0001).

CONCLUSIONS

Using a CGP assay capable of detecting all classes of GA simultaneously, an extraordinarily high frequency of CRGA was identified in a large series of patients with advanced UC. Cancer 2016;122:702-711. © 2015 American Cancer Society.

To understand the involvement of osteoclastogenesis inhibitory <em>factor</em> (OCIF), also called osteoprotegerin (OPG), in the pathogenesis of bone destruction in rheumatoid arthritis (RA), we investigated the cytokine network involved in the production of OCIF by human <em>fibroblast</em>-like synovial (HFLS) cells from a patient with RA. Inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 plus soluble IL-6 receptor (sIL-6R), IL-<em>17</em>, and tumor necrosis <em>factor</em> (TNF)-alpha, which are elevated in synovial fluid in RA, upregulated the production of OCIF to a level (5-20 ng/ml) sufficient to inhibit osteoclastogenesis in vitro. These inflammatory cytokines (except for IL-6 plus sIL-6R) stimulate OCIF production directly or indirectly through stimulation of prostaglandin E2 (PGE2) synthesis. In contrast to the findings with inflammatory cytokines, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) inhibited the production of OCIF by the cells in a dose-dependent manner. While bFGF enhanced both the inflammatory cytokine-mediated release of PGE2 and the PGE2-mediated OCIF production, it significantly suppressed OCIF production by negating the direct stimulatory effect of the inflammatory cytokines. These findings suggest that bFGF in the synovial fluid of patients with RA may lead to severe joint destruction by suppressing the production of OCIF by HFLS cells.

Prior to menopause, women have a lower risk of cardiovascular disease compared to age-matched men. Despite the well-documented beneficial physiological effects of ovarian hormones on vascular reactivity and growth, very little is known with regard to the direct action on cardiac cells.

OBJECTIVE

The following study examined the pattern of ovarian hormone receptor subtype expression in cardiac fibroblasts, the modulator role of 17 beta-estradiol and progesterone on growth and their respective influence on putative molecular events of extracellular matrix remodeling.

RESULTS

Neonatal rat cardiac fibroblasts were isolated from 1- to 3-day-old Sprague--Dawley rats. Immunofluorescence and Western blot analysis revealed the presence of estrogen receptor-alpha (ER-alpha), and -beta (ER-beta) subtypes, with the ER-alpha subtype localized on the plasma membrane. Likewise, both progesterone receptor-A (PR-A), and -B (PR-B) subtypes were expressed in cardiac fibroblasts, and the PR-B appeared to be the predominant subtype associated with the plasma membrane. Despite the presence of both ER subtypes, the treatment of cardiac fibroblasts with 1 microM 17 beta-estradiol exerted a modest decrease in DNA synthesis. By contrast, progesterone treatment caused a dose-dependent decrease in [3H]thymidine uptake, without a concomitant induction of apoptosis. The progesterone-mediated decrease in DNA synthesis was associated with the upregulation of the cyclin-dependent kinase inhibitor p27(Kip1), whereas p21(cip) and proliferating cell nuclear antigen protein levels were unchanged. Lastly, despite the modest effect on DNA synthesis, 17 beta-estradiol increased the steady-state mRNA levels of transforming growth factor-beta(3) and fibronectin. Likewise, progesterone increased the expression of both transforming growth factor-beta(3), and fibronectin mRNA.

CONCLUSIONS

Collectively, these data are the first to highlight the presence of estrogen and progesterone receptor subtypes on the plasma membrane of neonatal rat cardiac fibroblasts, and further underscore the ability of ovarian hormones to directly suppress DNA synthesis, and influence putative molecular events associated with extracellular matrix remodeling.

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) pathway acts in concert with the Frizzled homolog LIN-<em>17</em> to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

Analysis of <em>growth</em> regulation in B-chronic lymphocytic leukemia (B-CLL) is of pivotal importance for understanding the pathophysiology and the development of new therapeutic approaches. We investigated the effect of soluble ligands and the interaction with <em>fibroblasts</em> in an in vitro system developed for the expansion of normal B lymphocytes. A total of <em>17</em> peripheral blood and bone marrow samples from patients with untreated B-CLL were analyzed for survival, apoptosis, and bcl-2 protein expression. The most efficient stimulus for cell survival was cocultivation with CDw32-transfected murine <em>fibroblasts</em>, which achieved a median of 56% surviving CD5 positive B cells with a plateau between Day 3 and Day 13 (p < 0.0001). IL-4 alone had a significant, but less profound, effect on cell survival: cell viability was increased by a <em>factor</em> of 1.7 on Day 3 (p = 0.001), but cell viability continued to decline. In contrast, the soluble recombinant human CD40 ligand and two different anti-CD40 antibodies did not prolong cell survival. In all experiments prolongation of cell survival was accompanied by a significant reduction of apoptosis of the leukemic B cells: in CDw32-transfected <em>fibroblasts</em> apoptosis was reduced by a mean of 90%, in IL-4 by a mean of 55%. Reduction in apoptotic cell death was associated with elevated bcl-2 protein levels. Our results emphasize the critical role of the interaction between B-CLL cells and CDw32-transfected <em>fibroblasts</em> for cell viability in vitro. Prolongation of cell survival is caused by a reduction of apoptosis and correlates with bcl-2 protein expression.

Transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) is commonly used to promote matrix production for engineered tissues in vitro, yet it also enhances <em>fibroblast</em> contractility. For applications where contraction is undesirable, we hypothesized that epidermal <em>growth</em> <em>factor</em> (EGF) would yield equivalent mechanical properties without enhancing contractility. In this study, the response of human dermal <em>fibroblasts</em> to EGF (5 ng/mL) and TGF-beta1 (5 ng/mL) was determined within hemispheric fibrin-based gels by assessing matrix compaction and strength, cell number, collagen production, and contractility. After 3 weeks, both cytokines enhanced compaction relative to controls, and EGF roughly doubled matrix strength over controls and TGF-beta1-treated samples. TGF-beta1 induced alpha-smooth muscle actin (alphaSMA) expression whereas EGF did not. TGF-beta1 also increased retraction following substrate release while EGF reduced retraction. Treatment with cytochalasin D revealed that, regardless of <em>growth</em> <em>factor</em>, approximately 10% of the total retraction was due to residual matrix stress accumulated during cell-mediated remodeling. EGF increased the cell number by <em>17</em>%, whereas TGF-beta1 decreased the cell number by 63% relative to controls. EGF and TGF-beta1 stimulated greater collagen content than controls by 49% and 33%, respectively. These data suggest that EGF may be an attractive alternative to TGF-beta1 for engineering fibrin-based connective tissue substitutes with adequate strength and minimal tissue contractility.

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and <em>growth</em> <em>factors</em> to determine those <em>factors</em> that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> 1 (IGF1), and <em>fibroblast</em> <em>growth</em> <em>factor</em> did not influence DNA synthesis whereas transforming <em>growth</em> <em>factor</em> beta (TGF beta) alone had a significant effect. Neither estradiol-<em>17</em> beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal <em>growth</em> <em>factor</em> or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the <em>growth</em> promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell <em>growth</em> in addition to exerting pronounced effects on cytodifferentiation.

The present study was undertaken to investigate the effect of epidermal <em>growth</em> <em>factor</em> (EGF) on the biosynthetic activity of skin <em>fibroblasts</em> from donors of varying age and the modulation of their response to this <em>growth</em> <em>factor</em> by culture in a three-dimensional extracellular matrix. When cultured in monolayer on plastic or at the surface of a collagen gel, EGF specifically inhibited collagen synthesis whatever the age of the donor (from <em>17</em> to 84 years, n = 11). This inhibition was paralleled by a significant decrease in the steady-state level of procollagen type I mRNAs. When embedded in a three-dimensional floating collagen lattice, EGF stimulated the non-collagen protein (NCP) synthesis in <em>fibroblasts</em> from younger donors (5 out of 6) while <em>fibroblasts</em> from the older ones were not affected. Collagen production by <em>fibroblasts</em> from younger donors was not inhibited as in monolayer (some being even stimulated) while that of the older donors was inhibited as observed in monolayer. The steady-state level of procollagen type I mRNA was not modified by EGF in the three-dimensional culture. No significant difference was observed in the affinity and the number of EGF receptors of the <em>fibroblasts</em> on plastic or embedded in a collagen lattice between young and aged donors. Our results suggest that the environment of the cells can modulate the reactivity to EGF and reveal differences related to in vivo aging.

BACKGROUND

We previously reported the preliminary findings from a feasibility study of bladder cancer (BCa) screening with urinary molecular markers (Bladder Cancer Urine Marker Project [BLU-P]) that has now been terminated.

OBJECTIVE

To report the final results from BLU-P to determine whether mass screening for BCa is feasible and useful.

METHODS

BLU-P was a Dutch population-based study initiated in 2008 to evaluate BCa screening. A total of 6500 men were invited to participate in the study, 1984 (30.5%) agreed, and 1747 (88.1%) men completed the protocol and were followed for 2 yr.

METHODS

The screening protocol included home hematuria testing followed by molecular markers-nuclear matrix protein 22 (NMP22), microsatellite analysis (MA), fibroblast growth factor receptor 3 (FGFR3) mutation snapshot assay, and a custom methylation-specific (MLPA) test-to determine the need for cystoscopy.

METHODS

Outcomes included the number of cystoscopies and the cancer detection rate within and outside the protocol, as determined by linkage to national registries.

CONCLUSIONS

Overall, 409 men (23.4%) tested positive for hematuria and underwent molecular testing. Current smokers (n=295 [17%]) and past smokers (n=998 [58%]) were significantly more likely to test positive for hematuria than nonsmokers. Seventy-one of 75 men (94.6%) with positive molecular markers underwent the recommended cystoscopy. Four BCas and one kidney tumor were detected through this sequential protocol, whereas one BCa and one kidney tumor were missed through the screening program. Limitations include the possibility of healthy subject bias.

CONCLUSIONS

For BCa screening, use of a sequential protocol with home hematuria testing followed by molecular markers substantially reduced the number of cystoscopy recommendations compared with dipstick testing alone. A sequential screening approach may help minimize unnecessary invasive follow-up testing, with very few missed cancers. Nevertheless, this mass screening program had a very low diagnostic yield in an unselected asymptomatic European male population.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumour tissues and is of prognostic relevance in human malignancies such as renal cell carcinoma and leukaemia. This study presents the data of 104 serum samples of 20 patients suffering from breast cancer. Mean serum levels of bFGF in these patients were 13.9 +/- <em>17</em>. 1 (min 0, max 56.4) pg/ml and 2.4 +/- 5.9 (min 0, max 24.7) pg/ml, respectively (p = 0.01). Basic FGF reached a sensitivity of 61% at a specificity of 87% when applying a cut-off level of 5 pg/ml. A continuous increase of bFGF serum levels before the clinical detection of relapse (lead time) was seen in 3 out of 8 cases with a mean lead time of 4 months. Preoperative serum levels were not of prognostic value and showed no correlation with axillary lymph node metastasis. These preliminary results indicate that, in breast cancer patients, soluble bFGF may be useful in early detection of primary tumours, recurrences and monitoring of therapy.