THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

Insulin-associated changes in carnitine palmitoyltransferase activity are mediated through the insulin-like growth factor I pathway in the cultured neonatal rat cardiac myocyte

The mitochondrial carnitine palmitoyltransferase (CPT) system is composed of two proteins, CPT-I and CPT-II, involved in the transport of fatty acids into the mitochondrial matrix to undergo $\beta$-oxidation. CPT-I is located outside the inner membrane and CPT-II is located on the inner aspect of the inner membrane. The CPT proteins are distinct with different molecular weights and activities. The malonyl-CoA sensitivity of CPT-I has been proposed as a regulatory step in $\beta$-oxidation. Using the neonatal rat cardiac myocyte, assays were designed to discriminate between these activities in situ using digitonin and Triton X-100. With this methodology, we are able to determine the involvement of the IGF-I pathway in the insulin-mediated increase in CPT activities. Concentrations of digitonin up to 25 $\mu$M fail to release citrate synthase from the mitochondrial matrix or alter the malonyl-CoA sensitivity of CPT-I. If the mitochondrial matrix was exposed, malonyl-CoA insensitive CPT-II would reduce malonyl-CoA sensitivity. In contrast to digitonin, Triton X-100 (0.15%) releases citrate synthase from the matrix and exposes CPT-II. CPT-II activity is confirmed by the absence of malonyl-CoA sensitivity. To examine the effects of various agents on the expression and/or activity of CPT, it is necessary to use serum-free medium to eliminate mitogenic effects of serum proteins. Comparison of different media to optimize CPT activity and cell viability resulted in the decision to use Dulbecco's Modified Eagle medium supplemented with transferrin. In three established models of cardiac hypertrophy using the neonatal rat cardiac myocyte there is a significant increase in CPT-I and CPT-II activity in the treated cells. Analogous to the situation seen in the hypertrophy model, insulin also significantly increases the activity of the mitochondrial proteins CPT-I, CPT-II and cytochrome oxidase with a coinciding increase the expression of CPT-II and cytochrome oxidase mRNA. The removal of serum increases the I$\sb{50}$ (concentration of inhibitor that halves enzyme activity) of CPT-I for malonyl-CoA by four-fold. Incubation with insulin returns I$\sb{50}$ values to serum levels. Incubation with insulin significantly increases malonyl-CoA and ATP levels in the cells with a resulting reduction in palmitate oxidation. Once malonyl-CoA inhibition of CPT-I is removed by permeabilizing the cells, insulin significantly increases the oxidation of palmitoyl-CoA in a manner which parallels the increase in CPT-I activity. Interestingly, CPT-II activity increases significantly only at the tissue culture concentration (1.7 $\mu$M) of insulin suggesting that the IGF-I pathway may be involved. Supporting a role for the IGF-I pathway in the insulin-induced increase in CPT activity is the significant increase in the synthesis of both cellular and mitochondrial proteins as well as increased synthesis of CPT-II. Consistent with an IGF-mediated pathway for the effect of insulin, IGF-I (10 ng/ml) significantly increases the activities of both CPT-I and -II. An IGF-I analogue which inhibits the autophosphorylation of the IGF-I receptor blunts the insulin-mediated increase in CPT-I and -II activity by greater than 70% and virtually eliminates the IGF-I response by greater than 90%. This is the first study to demonstrate the involvement of the IGF-I pathway in the regulation of mitochondrial protein expression, e.g. CPT. ^

Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

The role of transforming growth factor beta signaling in cardiac hypertrophy and fibrosis in heart failure

Objective. To determine whether transforming growth factor beta (TGF-β) receptor blockade using an oral antagonist has an effect on cardiac myocyte size in the hearts of transgenic mice with a heart failure phenotype. ^ Methods. In this pilot experimental study, cardiac tissue sections from the hearts of transgenic mice overexpressing tumor necrosis factor (MHCsTNF mice) having a phenotype of heart failure and wild-type mice, treated with an orally available TGF-β receptor antagonist were stained with wheat germ agglutinin to delineate the myocyte cell membrane and imaged using fluorescence microscopy. Using MetaVue software, the cardiac myocyte circumference was traced and the cross sectional area (CSA) of individual myocytes were measured. Measurements were repeated at the epicardial, mid-myocardial and endocardial levels to ensure adequate sampling and to minimize the effect of regional variations in myocyte size. ANOVA testing with post-hoc pairwise comparisons was done to assess any difference between the drug-treated and diluent-treated groups. ^ Results. There were no statistically significant differences in the average myocyte CSA measured at the epicardial, mid-myocardial or endocardial levels between diluent treated littermate control mice, drug treated normal mice, diluent-treated transgenic mice and drug-treated transgenic mice. There was no difference between the average pan-myocardial cross sectional area between any of the four groups mentioned above. ^ Conclusions. TGF-β receptor blockade using oral TGF-β receptor antagonist does not alter myocyte size in MHCsTNF mice that have a phenotype of heart failure. ^

In vivo longitudinal studies of spinal cord injury and vascular endothelial growth factor treatment

Approximately 12,000 new cases of spinal cord injury (SCI) are added each year to the estimated 259,000 Americans living with SCI. The majority of these patients return to society, their lives forever changed by permanent loss of sensory and motor function. While there are no FDA approved drugs for the treatment of SCI or a universally accepted standard therapy, the current though controversial treatment includes the delivery of high dosages of the corticosteroid methyliprednisolone sodium succinate, surgical interventions to stabilize the spinal column, and physical rehabilitation. It is therefore critically important to fully understand the pathology of injury and determine novel courses and rationally-based therapies for SCI. ^ Vascular endothelial growth factor (VEGF) is an attractive target for treating central nervous system (CNS) injury and disease because it has been shown to influence angiogenesis and neuroprotection. Preliminary studies have indicated that increased vasculature may be associated with functional recovery; therefore exogenous delivery of a pro-angiogenic growth factor such as VEGF may improve neurobehavioral outcome. In addition, VEGF may provide protection from secondary injury and result in increased survival and axonal sprouting. ^ In these studies, SCI rats received acute intraspinal injections of VEGF, the antibody to VEGF, or vehicle control. The effect of these various agents was investigated using longitudinalmulti-modal magnetic resonance imaging (MRI), neuro- and sensory behavioral assays, and end point immunohistochemistry. We found that rats that received VEGF after SCI had increased tissue sparing and improved white matter integrity at the earlier time points as shown by advanced magnetic resonance imaging (MRI) techniques. However, these favorable effects of VEGF were not maintained, suggesting that additional treatments with VEGF at multiple time points may be more beneficial, Histological examinations revealed that VEGF treatment may result in increased oligodendrogenesis and therefore may eventually lead to remyelination and improved functional outcome. ^ On the neurobehavioral studies, treatments with VEGF and Anti-VEGF did not significantly affect performance on tests of open-field locomotion, grid walk, inclined plane, or rearing. However, VEGF treatment resulted in significantly increased incidence of chronic neuropathic pain. This phenomenon could possibly be attributed to the fact that VEGF treatment may promote axonal sprouting and also results in tissue sparing, thereby providing a substrate for the growth of new axons. New connections made by these sprouting axons may involve components of pathways involved in the transmission of pain and therefore result in increased pain in those animals. ^

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Regulation of transforming growth factor β- and activin-induced transcription by mammalian Mad proteins

Members of the transforming growth factor β (TGF-β) superfamily are involved in diverse physiological activities including development, tissue repair, hormone regulation, bone formation, cell growth, and differentiation. At the cellular level, these functions are initiated by the interaction of ligands with specific transmembrane receptors with intrinsic serine/threonine kinase activity. The signaling pathway that links receptor activation to the transcriptional regulation of the target genes is largely unknown. Recent work in Drosophila and Xenopus signaling suggested that Mad (Mothers against dpp) functions downstream of the receptors of the TGF-β family. Mammalian Mad1 has been reported to respond to bone morphogenetic protein (BMP), but not to TGF-β or activin. We report here the cloning and functional studies of a novel mammalian Mad molecule, Mad3, as well as a rat Mad1 homologue. Overexpression of Mad3 in a variety of cells stimulated basal transcriptional activity of the TGF-β/activin-responsive reporter construct, p3TP-Lux. Furthermore, expression of Mad3 could potentiate the TGF-β- and activin-induced transcriptional stimulation of p3TP-Lux. By contrast, overexpression of Mad1 inhibited the basal as well as the TGF-β/activin induced p3TP-Lux activity. These findings, therefore, support the hypothesis that Mad3 may serve as a mediator linking TGF-β/activin receptors to transcriptional regulation.

Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus

Retroviral elements are found in abundance throughout the human genome but only rarely have alterations of endogenous genes by retroviral insertions been described. Herein we report that a human endogenous retrovirus (HERV) type C is inserted in the human growth factor gene pleiotrophin (PTN) between the 5′ untranslated and the coding region. This insert in the human genome expands the region relative to the murine gene. Studies with promoter-reporter constructs show that the HERV insert in the human PTN gene generates an additional promoter with trophoblast-specific activity. Due to this promoter function, fusion transcripts between HERV and the open reading frame of PTN (HERV-PTN) were detected in all normal human trophoblast cell cultures as early as 9 weeks after gestation (n = 7) and in all term placenta tissues (n = 5) but not in other normal adult tissues. Furthermore, only trophoblast-derived choriocarcinoma cell lines expressed HERV-PTN mRNA whereas tumor cell lines derived from the embryoblast (teratocarcinoma) or from other lineages failed to do so. We investigated the significance of HERV-PTN mRNA in a choriocarcinoma model by targeting this transcript with ribozymes and found that the depletion of HERV-PTN mRNA prevents human choriocarcinoma growth, invasion, and angiogenesis in mice. This suggests that the tissue-specific expression of PTN due to the HERV insertion in the human genome supports the highly aggressive growth of human choriocarcinoma and possibly of the human trophoblast.