<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are known to play an important role in the <em>growth</em> of normal prostatic epithelial cells. In addition to their effects on proliferation, FGFs can promote cell motility, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGFs are overexpressed in human prostate cancers, we analyzed 26 prostate cancer RNAs by reverse transcription-PCR for expression of FGF3, FGF4, and FGF6, which cannot be detected in normal prostate tissue by this technique. Fourteen of 26 prostate cancers expressed FGF6 mRNA. No expression of FGF3 or FGF4 was detected. An ELISA of tissue extracts of normal prostate, high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer for FGF6 showed that this <em>growth</em> <em>factor</em> was undetectable in normal prostate but was present at elevated levels in 4 of 9 PIN lesions and in <em>15</em> of 24 prostate cancers. Immunohistochemical analysis with anti-FGF6 antibody revealed weak staining of prostatic basal cells in normal prostate that was markedly elevated in PIN. In the prostate cancers, the majority of cases revealed expression of FGF6 by the prostate cancer cells themselves. In two cases, expression was present in prostatic stromal cells. Exogenous FGF6 was able to stimulate proliferation of primary prostatic epithelial and stromal cells, immortalized prostatic epithelial cells, and prostate cancer cell lines in tissue culture. FGF receptor 4, which is the most potent FGF receptor for FGF6, is expressed in the human prostate in vivo and in all of the cultured cell lines. Thus, FGF6 is increased in PIN and prostate cancer and can promote the proliferation of the transformed prostatic epithelial cells via paracrine and autocrine mechanisms.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor (FGFR) substrate 2 (FRS2) is an adaptor protein that plays a critical role in FGFR signaling. FRS2 is located on chromosome 12q13-<em>15</em> that is frequently amplified in liposarcomas. The significance of FRS2 and FGFR signaling in high-grade liposarcomas is unknown. Herein, we first comparatively examined the amplification and expression of FRS2 with CDK4 and MDM2 in dedifferentiated liposarcoma (DDLS) and undifferentiated high-grade pleomorphic sarcoma (UHGPS). Amplification and expression of the three genes were identified in 90% to 100% (9-11 of 11) of DDLS, whereas that of FRS2, CDK4, and MDM2 were observed in 55% (41 of 75), 48% (36 of 75), and 44% (33/75) of clinically diagnosed UHGPS, suggesting that these "UHGPS" may represent DDLS despite lacking histologic evidence of lipoblasts. Immunohistochemical analysis of phosphorylated FRS2 protein indicated that the FGFR/FRS2 signaling axis was generally activated in about 75% of FRS2-positive high-grade liposarcomas. Moreover, we found that FRS2 and FGFRs proteins are highly expressed and functional in three high-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2, and SW872. Importantly, the FGFR selective inhibitor NVP-BGJ-398 significantly inhibited the <em>growth</em> of FU-DDLS-1 and LiSa-2 cells with a concomitant suppression of FGFR signal transduction. Attenuation of FRS2 protein in FU-DDLS-1 and LiSa-2 cell lines decreased the phosphorylated extracellular signal-regulated kinase 1/2 and AKT and repressed cell proliferation. These findings indicate that analysis of FRS2 in combination with CDK4 and MDM2 will more accurately characterize pathologic features of high-grade liposarcomas. Activated FGFR/FRS2 signaling may play a functional role in the development of high-grade liposarcomas, therefore, serve as a potential therapeutic target.

OBJECTIVE

To examine whether fetal human retinal cells can be maintained in vitro over long time periods and to determine whether exogenous growth factors can be used to generate large numbers of photoreceptors within these cultures.

METHODS

Fetal human retinas (6 to 13 weeks after conception) were dissected, dissociated, and plated into culture wells. Specific growth factors and steroid/thyroid hormones, which have been shown to influence retinal progenitor cell proliferation and differentiation in rats, were added to the culture medium to determine whether any of these factors had similar effects on human retinal cells.

RESULTS

Fetal human retinal cells survived and continued to proliferate for up to 300 days in vitro. Under control conditions, 15 million cells were generated from an initial plating of 100,000 cells; however, the addition of either epidermal growth factor or basic fibroblast growth factor stimulated proliferation and resulted in the generation of more than 100 million cells. A percentage of these cells was induced to differentiate as photoreceptors by adding either retinoic acid or triiodo-thyronine to the culture medium.

CONCLUSIONS

Fetal human retinal cells can be maintained and expanded in vitro, indicating that this technique may be useful for generating large numbers of retinal cells. The number and types of cells generated can be influenced by adding exogenous factors to the culture medium. The response of human retinal cells to growth factors and hormones is similar to the response of rodent retinal cells to the same factors, suggesting that the effects of these factors are conserved across species.

In cell populations exposed to ionizing radiation, the biological effects occur in a much larger proportion of cells than are estimated to be traversed by radiation. It has been suggested that irradiated cells are capable of providing signals to the neighboring unirradiated cells resulting in damage to these cells. This phenomenon is termed the bystander effect. The bystander effect induces persistent, long-term, transmissible changes that result in delayed death and neoplastic transformation. Because the bystander effect is relevant to carcinogenesis, it could have significant implications for risk estimation for radiation exposure. The nature of the bystander effect signal and how it impacts the unirradiated cells remains to be elucidated. Examination of the changes in gene expression could provide clues to understanding the bystander effect and could define the signaling pathways involved in sustaining damage to these cells. The microarray technology serves as a tool to gain insight into the molecular pathways leading to bystander effect. Using medium from irradiated normal human diploid lung <em>fibroblasts</em> as a model system we examined gene expression alterations in bystander cells. The microarray data revealed that the radiation-induced gene expression profile in irradiated cells is different from unirradiated bystander cells suggesting that the pathways leading to biological effects in the bystander cells are different from the directly irradiated cells. The genes known to be responsive to ionizing radiation were observed in irradiated cells. Several genes were upregulated in cells receiving media from irradiated cells. Surprisingly no genes were found to be downregulated in these cells. A number of genes belonging to extracellular signaling, <em>growth</em> <em>factors</em> and several receptors were identified in bystander cells. Interestingly <em>15</em> genes involved in the cell communication processes were found to be upregulated. The induction of receptors and the cell communication processes in bystander cells receiving media from irradiated cells supports the active involvement of these processes in inducing bystander effect.

The human proto-oncogene INT2 (homologous to the mouse INT2 gene, implicated in proviral induced mammary carcinoma) has been mapped to chromosome 11q13 and found to share band localisation with, among others, the HST1 proto-oncogene. Both genes are members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. In the present study, coamplification (2-<em>15</em> copies) of the INT2/HST1 genes was found in 27 (9%) of 311 invasive human breast carcinomas using slot blot and Southern blot analyses. Amplification was not correlated to tumour size, axillary lymph node status or stage of disease, neither to patient age nor menopausal status. However, 26 (96%) of the 27 amplified tumours were, often strongly, Oestrogen receptor positive compared to 65% of the unamplified cases (P = 0.001). These findings are in sharp contrast to the strong correlations of HER-2/neu proto-oncogene amplification with advanced stage and steroid receptor negativity, previously observed in the same series of tumours. Patients with INT2/HST1 amplified breast cancer had a significantly shorter disease-free survival compared to those with unamplified genes (P = 0.0<em>15</em>, median follow up 45 months). This correlation was confined to node-negative patients and persisted in multivariate analysis. No significant correlation to survival from breast cancer was found. It is concluded that amplification of the 11q13 region in breast cancer occurs in a particular subset of aggressive tumours, quite different from that identified by HER-2/neu amplification. It still remains to be shown that the selection for amplified genes at 11q13 is due to the activity of INT2, HST1 or yet another, still unidentified, neighbouring gene. However, the results are potentially of clinical value in separating a group of node-negative breast cancer for more intense treatment.

Acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> are heparin-binding proteins that induce cellular proliferation, mesodermal development, and vascular <em>growth</em>. As such, they may be important in cardiac development and disease. To determine whether cardiac myocytes contain <em>fibroblast</em> <em>growth</em> <em>factors</em>, neonatal rat cardiac myocytes were studied in primary culture and compared to primary cultures of nonmyocyte cardiac cells. Northern blot analysis revealed a 4.0-kilobase mRNA in myocytes that hybridized to acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> cDNA and was not detectable in nonmyocyte cultures. Western blot analysis demonstrated the accumulation of a <em>15</em>-kDa peptide with immunological identity to acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> in extracts of extracellular matrix from myocyte cultures that was not detectable in similar extracts of nonmyocyte extracellular matrix. No acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>-like protein was detectable in cellular lysates from either myocyte or nonmyocyte cultures. These results demonstrate that neonatal cardiac myocytes express acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA and deposit a protein with immunological identity to acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> into the extracellular matrix. The results suggest that acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> produced by cardiac myocytes may mediate, through both paracrine and autocrine mechanisms, such diverse processes as myocyte differentiation, cellular proliferation, and vascular <em>growth</em> in the heart.

A cell-free assay has been developed to detect and characterize a nerve <em>growth</em> <em>factor</em> (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches <em>15</em>-20-fold; activation is nearly as high with <em>fibroblast</em> <em>growth</em> <em>factor</em>, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal <em>growth</em> <em>factor</em> and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for <em>growth</em>-<em>factor</em>-promoted neuronal differentiation.

Epidermal <em>growth</em> <em>factor</em> (EGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and nerve <em>growth</em> <em>factor</em> (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGF-stimulated PC12 cells by an anti-phosphotyrosine antibody. All three <em>growth</em> <em>factors</em> increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is <em>15</em>- to 20-fold, 2- to 3-fold, and 8- to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-gamma to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these <em>growth</em> <em>factors</em>. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite out<em>growth</em> but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these <em>growth</em> <em>factors</em>, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.

PURPOSE: The association of chemotherapy and antiangiogenic drugs has shown efficacy in clinical oncology. However, there is a need for biomarkers that allow selection of patients who are likely to benefit from such treatment and are useful for indicating best drug combination and schedule. EXPERIMENTAL DESIGN: We investigated the predictive potential of six angiogenic molecules/transcripts and nine subpopulations of circulating endothelial cells (CEC) and progenitors (CEP) in 46 patients with advanced breast cancer treated with metronomic cyclophosphamide and capecitabine plus bevacizumab. RESULTS: Median time to progression was 281 days. Baseline CECs higher than the first quartile were associated with an increased time to progression (P = 0.021). At progression, CECs were markedly reduced (P = 0.0002). In the cohort of <em>15</em> long-term responders, who progressed later than 1 year after beginning of therapy, circulating vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A levels measured after 2 months of therapy were significantly reduced, and there were significant trends toward lower levels of PDGF-BB, CEPs, and CECs. At the time of progression, angiogenic <em>growth</em> <em>factors</em> VEGF-A and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were significantly increased. CONCLUSIONS: Baseline CECs (likely reflecting an active vascular turnover) predicted a prolonged clinical benefit. At the time of relapse, a pattern of decreased CECs and increased angiogenic <em>growth</em> <em>factors</em> suggested a switch toward a different type of cancer vascularization. VEGF-A and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels after 2 months of therapy were also useful to identify patients whose disease was likely to progress. These biomarkers are likely to be useful for treatment selection and might be incorporated in design of future studies. (Clin Cancer Res 2009;<em>15</em>(24):7652-7).

c-Src non-receptor tyrosine kinase is an important component of the platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in <em>fibroblasts</em>. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src-family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining <em>15</em> proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates - Calpain 2, Eps<em>15</em> and Trim28 - in a c-Src-dependent fashion.

OBJECTIVE

To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro.

METHODS

Primary CLPF cultures were incubated with TGF-beta 1 and analyzed for production of alpha-smooth muscle actin (alpha-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration.

RESULTS

Incubation of CLPF with TGF-beta 1 for 2 d did not change alpha-SMA levels, while TGF-beta 1 treatment for 6 d significantly increased alpha-SMA production. Short term incubation (6 h) with TGF-beta 1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-beta 1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-beta 1 (2 d) in contrast to long term incubation with TGF-beta 1 for 6 d. After induction of migration, TGF-beta 1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.

CONCLUSIONS

Long term incubation of CLPF with TGF-beta 1 induced differentiation into myofibroblasts with enhanced alpha-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-beta 1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of alpha-SMA production.

OBJECTIVE

Chronic atrial fibrillation (AF) is characterized by a remodeling process which involves the development of fibrosis. Since angiotensin II has been suspected to be involved in this process, the aim of our study was to investigate a possible influence of an ACE-I therapy in patients with chronic AF regarding the occurrence of left atrial structural remodeling.

METHODS

Atrial tissue samples were obtained from patients with lone chronic AF or sinus rhythm (SR). Collagen I, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein expression were measured by quantitative Western Blotting techniques and calculated as mean +/- SEM. Histological tissue samples were used for calculating microvessel density (microvessel/mm(2) +/- SEM).

RESULTS

In AF, the collagen amount was higher (1.78 +/- 0.21; p = 0.01) vs. SR (0.37 +/- 0.07) accompanied by declining microcapillary density (AF: 145 +/- 13 vs. SR: 202 +/- 9; p = 0.01). Additionally, a negative correlation (p = 0.01) between collagen content and microcapillary density was observed. To investigate the influence of an ACE-I therapy on this remodeling process, patient groups were divided into AF and SR both with or without ACE-I. Interestingly, there was a significantly lower expression of collagen I in AF with ACE-I (1.04 +/- 0.26) vs. AF without ACE-I treatment (2.07 +/- 0.24, p = 0.02). The microcapillaries were not diminished in AF with ACE-I (180 +/- 15) vs. SR with ACE-I (196 +/- 9), but there was a significant rarification in AF without ACE-I (123 +/- 18; p = 0.03). The expression of VEGF and bFGF did not reveal any significant differences.

CONCLUSIONS

In patients undergoing ACE-I treatment: atrial structural remodeling was attenuated and the loss of atrial microcapillaries was prevented.

Human umbilical vein endothelial cells were cultured in supernatants of peripheral blood monocytes that had been cultured for 3 days with and without lactoferrin. Colony-stimulating activity (CSA) was measured in supernatants of the endothelial cell cultures and appropriate control cultures using normal, T-lymphocyte-depleted, phagocyte-depleted, low-density bone marrow cells in colony <em>growth</em> (CFU-GM) assays. Monocyte-conditioned medium contained a nondialyzable, heat labile <em>factor</em> that enhanced 4-<em>15</em>--fold the production of CSA by endothelial cells. The addition of lactoferrin to monocyte cultures reduced the activity of this monokine by 69%. Lactoferrin did not inhibit CSA production by monokine-stimulated endothelial cells. Therefore, vascular endothelial cells are potent sources of CSA, the production of CSA by these cells is regulated by a stimulatory monokine, and the production and/or release of the monokine is inhibited by lactoferrin, a neutrophil-derived putative feedback inhibitor of granulopoiesis. Inasmuch as a similar monokine is known to stimulate CSA production by <em>fibroblasts</em> and T lymphocytes, we suggest that mononuclear phagocytes play a pivotal role in the regulation of granulopoiesis by recruiting a variety of cell types to produce CSA.

BACKGROUND

Mutations in fibroblast growth factor 3 receptor (FGFR3) are frequent events in low-grade bladder tumors. To assess the potential utility of the detection of FGFR3 mutations in a screening modality, the authors analyzed urine sediment DNA samples from 192 patients in a retrospective study.

METHODS

Urine sediment DNA samples from 192 patients were prepared. Seventy-two patients had undergone transurethral resection (TURBT group) of mainly Ta lesions and 120 patients had undergone cystectomy (cystectomy group). The majority of patients in the cystectomy group had more advanced tumors compared with patients in the TURBT group. DNA preparations were screened for FGFR3 mutations in exons 7, 10, and 15 using single-strand conformation polymorphism (SSCP) and DNA sequencing.

RESULTS

Using SSCP, 67% of patients in the TURBT group and 28% in the cystectomy group displayed FGFR3 mutations. Comparative analysis of cytology results and FGFR3 mutational analysis were performed in 122 cases. Within the TURBT group, FGFR3 mutation analysis outperformed cytology. FGFR3 mutation analysis identified change in 68% of urine sediment DNA samples whereas cytology recorded the presence of tumor cells in 32% of the DNA samples. In the cystectomy group, cytology outperformed FGFR3 mutation analysis. Cytology recorded tumor detection in 90% of patients, while SSCP identified mutational change in 24%.

CONCLUSIONS

Combining FGFR3 mutation results with cytology in both groups correctly identified tumor presence in 105 of 122 (86%) of patients. The greater sensitivity of FGFR3 mutation detection over cytology in identifying the presence of low-grade, superficial bladder tumors represents a potential new tool to complement standard cytology in screening patients for bladder tumors and recurrent disease.

The translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction in IIC9 <em>fibroblasts</em> has been studied to define the functions of 1,2-diacylglycerol (DAG) derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). alpha-Thrombin caused a biphasic change in DAG, with two peaks at <em>15</em>-60 s and 5-<em>15</em> min, derived from PIP2 and PC, respectively, while platelet-derived <em>growth</em> <em>factor</em> (PDGF) induced a monophasic DAG increase from PC at 5-<em>15</em> min. alpha-Thrombin also induced a rapid, but transient, increase of inositol 1,4,5-trisphosphate and cytosolic Ca2+, whereas PDGF did not. Three PKC isozymes, alpha, epsilon, and zeta, were identified by Western blotting in IIC9 cells and were mainly localized in the cytosol. A fraction of cytosolic PKC alpha was rapidly translocated by alpha-thrombin at <em>15</em> s, but its membrane association was lost within 1 min. PKC epsilon was also rapidly translocated; however, its membrane association was sustained for almost 60 min. PKC zeta was not translocated by alpha-thrombin or phorbol 12-myristate 13-acetate. PDGF translocated PKC epsilon at 5 min but had little effect at <em>15</em> s and did not translocate PKC alpha or zeta. Incubation with Bacillus cereus PC- or phosphatidylinositol-specific phospholipase C, which increased DAG but not phosphatidic acid, stimulated translocation of PKC epsilon, but not PKC alpha or zeta. Addition of chelators to inhibit the rise in intracellular Ca2+ largely blocked PKC alpha translocation induced by alpha-thrombin but had no effect on PKC epsilon translocation. Addition of ionomycin allowed alpha-thrombin to induce PKC alpha translocation at 5 min. PKC alpha translocation was mimicked by 1,2-dioctanoylglycerol plus ionomycin, but not by either alone. On the other hand, PKC epsilon was translocated by the DAG alone. These results support the conclusion that PIP2 hydrolysis activates both PKC alpha and epsilon at <em>15</em> s, whereas PC hydrolysis activates only PKC epsilon at 5 min. The differential activation at 5 min can be attributed to the failure of PC hydrolysis to increase Ca2+ and not to a difference in the molecular species of DAG derived from the phospholipids.

OBJECTIVE

We have shown that basic fibroblast growth factor (bFGF/FGF-2) enhances myocardial collateral development in a canine model of progressive coronary occlusion when delivered via the left atrial or intracoronary routes; however, we have found intravenous bFGF ineffective in the same model. Data on the fate and efficacy of intravenous bFGF are limited. We hypothesized that first pass lung uptake might limit myocardial bFGF availability after intravenous injection. We postulated that delivery of bFGF through the distal port of a wedged Swan Ganz catheter might circumvent this problem by restricting exposure of bFGF to a limited number of pulmonary binding sites. This study evaluated differential regional uptake of 125I labeled bFGF following bolus intravenous, Swan Ganz, left atrial, intracoronary, and pericardial delivery.

METHODS

Mongrel dogs were used. Human recombinant bFGF, monoiodinated with 125I, was mixed with cold bFGF to a specific activity of 0.03 microCi/microgram. Approximately 100 micrograms/kg was injected per animal by the intravenous, left atrial, Swan Ganz, intracoronary, or pericardial route. Dogs were killed 15 min or 150 min later. The heart, lungs, liver, spleen, and kidneys were harvested and 125I activity was assessed. Immunohistochemical and pharmacokinetic studies were also performed.

RESULTS

Serum half life of bFGF was comparable after intracoronary, intravenous and left atrial delivery (50 min); however, there were significant differences with regard to pharmacodynamics. After intracoronary administration, 3-5% of the total bFGF dose was recovered from the heart, with the peptide immunolocalized to the extracellular matrix and vascular endothelium. In contrast, only 1.3% of the injected bFGF was localized to the heart after left atrial administration and 0.5% was recovered after intravenous or Swan Ganz delivery. Pericardial administration resulted in substantial cardiac bFGF delivery; 19% was present at 150 min. Myocardial uptake was similar with Swan Ganz and intravenous delivery, suggesting that the administered dose did not saturate available pulmonary binding sites.

CONCLUSIONS

These data predict efficacy of intracoronary, left atrial, and pericardial bFGF for myocardial angiogenesis, and a lack of efficacy after bolus intravenous and Swan Ganz administration.

beta-Klotho, a newly described membrane protein, regulates bile acid synthesis. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>15</em> (FGF-<em>15</em>) and FGF receptor-4 (FGFR4) knockout mice share a similar phenotype with beta-Klotho-deficient mice. FGF-<em>15</em> secretion by the intestine regulates hepatic bile acid biosynthesis. The effects of beta-Klotho and FGF-<em>15</em> on the ileal apical sodium bile transporter (ASBT) are unknown. beta-Klotho siRNA treatment of the mouse colon cancer cell line, CT-26, and the human intrahepatic biliary epithelial cells (HIBEC) resulted in upregulation of endogenous ASBT expression that was associated with reduced expression of the farnesoid X receptor (FXR) and the short heterodimer partner (SHP). Silencing beta-Klotho activated the ASBT promoter in CT-26, Mz-ChA-1 (human cholangiocarcinoma), and HIBEC cells. Site-directed mutagenesis of liver receptor homolog-1 (mouse) or retinoic acid receptor/retinoid X receptor (RAR/RXR) (human) cis-elements attenuated the basal activity of the ASBT promoter and abrogated its response to beta-Klotho silencing. siSHP, siFXR, or dominant-negative FXR treatment also eliminated the beta-Klotho response. FGF-<em>15</em> secretion into cell culture media by CT-26 cells was diminished after siFGF-<em>15</em> or sibeta-Klotho treatment and enhanced by chenodeoxycholic acid. Exogenous FGF-19 repressed ASBT protein expression in mouse ileum, gallbladder, and in HIBEC and repressed ASBT promoter activity in Caco-2, HIBEC, and Mz-ChA-1 cells. Promoter repression was dependent on the expression of FGFR4. These results indicate that both beta-Klotho and FGF-<em>15</em>/19 repress ASBT in enterocytes and cholangiocytes. These novel signaling pathways need to be considered in analyzing bile acid homeostasis.

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription <em>factor</em> and plays an important role in <em>growth</em>, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial <em>fibroblasts</em>. PPARgamma ligands <em>15</em>-deoxy-Delta(12,14) prostaglandin J(2) (<em>15</em>d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. <em>15</em>d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both <em>15</em>d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic <em>factor</em>, and/or vascular endothelial <em>growth</em> <em>factor</em> (VEGF), a potent angiogenic <em>factor</em>, comparing the relationships with those of high- and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP-1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n = 11) than in HCC with high vascularity (n = <em>15</em>). In contrast, T/N ratios of VEGF mRNA level in CCC (n = 11) were comparable with those in HCC with low vascularity (n = 5). In CCC, the cancer cells and <em>fibroblasts</em> were positively stained with anti-TSP-1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP-1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP-1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP-1 in CCC or VEGF in HCC.

Genomic imprinting is a novel form of control of gene expression in which the transcription of each allele of an imprinted gene is dependent on the sex of the gamete from which it was derived; to date>> <em>15</em> genes have been demonstrated to show imprinting. The maintenance of a normal imprinting pattern in many loci has been shown to be essential for normal development and adult life. Many tumours, and some developmental disorders, exhibit loss of imprinting (LOI) in key genes such as insulin-like <em>growth</em> <em>factor</em> 2 (IGF2) which often results in hyperplasia and is associated with cancer. The mechanism by which the genomic imprint is first established, then maintained, is not understood. However, in the case of IGF2, the expression of a neighbouring gene, H19, has been suggested to influence its transcription by competition for a common enhancer, thereby generating a mutually exclusive and allele-specific pattern of gene expression. Associated changes in CpG methylation in discrete areas of both genes have been implicated in maintenance of the imprint. We have examined the allele-specific expression of IGF2 and H19 in <em>fibroblasts</em> derived from patients with sporadic Beckwith-Wiedemann syndrome (BWS), a fetal over<em>growth</em> syndrome associated with an imprinted locus on 11p<em>15</em>.5. We report that the majority of karyotypically normal patients show LOI of IGF2 with biallelic expression. In a proportion of these patients, loss of IGF2 imprinting was associated with complete suppression of H19 expression, as predicted by the enhancer competition model. However, in a significant number of cases, IGF2 showed biallelic expression even though H19 expression and methylation status were normal. This indicates that there must be an alternative H19-independent pathway by which allele-specific IGF2 expression is established or maintained.

Dysregulation of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) by the translocation t(4;14)(p16;q32) occurs in <em>15</em>% of multiple myeloma (MM) patients and confers a <em>growth</em> and survival advantage to malignant plasma cells. As FGFR3 is a molecular target, we assessed the therapeutic potential of the FGFR-specific tyrosine kinase inhibitors SU5402 and SU10991 in MM. SU5402 inhibited FGFR3 phosphorylation in vitro and in murine MM tumour models. B cells dependent on FGFR3 for survival were specifically sensitive to SU5402. A panel of 11 human myeloma cell lines was studied, five bearing the t(4;14) translocation. The KMS11 human myeloma cell line, which expresses constitutively active mutant FGFR3, displayed an 85% decrease in S-phase cells, a 95% increase in G0/G1 cells, and 4.5-fold increase in apoptotic cells after 72 h treatment with 10 micromol/l SU5402. Activated extracellular signal-regulated kinases 1 and 2 and signal transducer and activator of transcription 3 were rapidly down-regulated after SU5402 treatment. In human myeloma cell lines expressing wild-type FGFR3 the stimulating effect of aFGF ligand was abrogated by SU5402 treatment. Myeloma cells lacking the t(4;14) or with the t(4;14) and a secondary RAS mutation did not respond to therapy. These findings support the development of clinical trials of early intervention with FGFR3 inhibitors in t(4;14) myeloma.

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic <em>fibroblast</em> (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -<em>15</em>, and -9, respectively) exhibited typical hES cells characteristics, including continuous <em>growth</em>, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic <em>factors</em> and extracellular matrices by hUECs may be important to the efficient <em>growth</em> of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.

OBJECTIVE

To examine for the expression of <em>15</em>-lipoxygenase 1 (<em>15</em>-LOX1) and <em>15</em>-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis.

METHODS

Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine <em>15</em>-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in <em>15</em>(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis.

RESULTS

HRMVECs expressed both <em>15</em>-LOX1 and <em>15</em>-LOX2. Hypoxia induced the expression of <em>15</em>-LOX1 and the production of its arachidonic acid metabolites <em>15</em>(S)-hydroxyeicosatetraenoic acid (<em>15</em>(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). <em>15</em>(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, <em>15</em>(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated <em>15</em>(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed <em>15</em>(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated <em>15</em>(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked <em>15</em>(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis.

CONCLUSIONS

These results suggest that hypoxia, through the induction of <em>15</em>-LOX1 expression, leads to the production of <em>15</em>(S)-HETE in HRMVECs. In addition, <em>15</em>(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.

OBJECTIVE

The purpose is to analyze the possible correlation between expression and activation of the high-affinity nerve growth factor (NGF) receptor TrkA, cell cycle protein expression, and disease outcome in serous ovarian carcinoma. In addition, we wished to study the possible link between expression of NGF, a novel angiogenic factor and its receptor TrkA, and the expression of factors involved in angiogenesis in effusions and solid tumors.

METHODS

Sections from 80 malignant effusions and 65 corresponding solid tumors were evaluated for protein expression of NGF, TrkA, and phospho-TrkA (p-TrkA). Effusions were additionally studied for expression of p53, p21(WAF1/CIP1), Ki-67, and the M(r) 85,000-cleaved fragment of poly(ADP-ribose) polymerase (p85-PARP) using immunohistochemistry (IHC). Thirty-two effusions were studied for TrkA, p-TrkA, p53, and p21(WAF1/CIP1) expression using immunoblotting. mRNA expression of basic fibroblast growth factor (bFGF), interleukin 8, and vascular endothelial growth factor (VEGF) was studied in 63 effusions and all solid tumors using in situ hybridization. Protein expression of bFGF, interleukin 8, and VEGF was additionally studied in 30 effusions using IHC.

RESULTS

NGF, TrkA, and p-TrkA were expressed in carcinoma cells in effusions in 60 of 80 (75%), 64 of 80 (80%), and 15 of 80 (19%) specimens, respectively. In solid tumors, p-TrkA expression was more frequent (52 of 65 tumors; 80%) and was accompanied by p-TrkA expression in endothelial cells. NGF colocalized with bFGF protein (P = 0.016) and mRNA (P = 0.032) in effusions, and with VEGF (P < 0.001) and bFGF (P = 0.008) in solid tumors. In survival analysis, expression of p85-PARP (P = 0.017) and cytoplasmic TrkA (P < 0.001) in effusions predicted better outcome, whereas membrane expression of p-TrkA in solid tumors correlated with poor survival (P = 0.004). Diffuse expression of p53 and Ki-67 was often seen using IHC, whereas p21(WAF1/CIP1) and p85-PARP expression was infrequent and focal. None of these correlated with NGF or TrkA expression or activity.

CONCLUSIONS

Coexpression of NGF with molecules involved in angiogenesis and p-TrkA expression in endothelial cells suggest that the proangiogenic role attributed to NGF in vitro and in vivo may be relevant in clinical cancer. Expression of p85-PARP as a marker of apoptosis and cytoplasmic expression of TrkA (probably representing nonglycosylated receptor) predict better outcome, whereas p-TrkA activation correlates with poor outcome in advanced stage serous ovarian carcinoma.