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• W4211072571 abstract "Acta OphthalmologicaVolume 86, Issue thesis4 p. 7-19 Free Access Introduction: Uveal melanoma First published: 19 November 2008 https://doi.org/10.1111/j.1755-3768.2008.01186.x Mario A. EconomouSt Eziks Eye HospitalKarolinska InstitutePolhemsgatan 50, SE 112 82 Stockholm, Sweden Tel: +4686723218Fax: +4686723375Email: mario-alexander.economou@sanktezik.se AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Anatomy, Histopathology, Epidemiology The iris, ciliary body and choroid constitute the uveal tract. The uveal tract is embryologically derived from the mesoderm and neural crest. It is a highly vascularized structure composed of melanocytes, supporting connective tissue with fibroblasts and blood vessels and peripheral nerves. Histopathologically there are four different types of uveal melanoma (UM) identified: spindle cell, epithelioid, mixed and necrotic. Approximately 5% of all UM arise in ocular and adnexal structures (Chang et al. 1998). Most ocular melanomas (85%) are uveal in origin, whereas primary conjuctival and orbital melanomas are very rare (Chang et al. 1998; Singh & Topham 2003). Approximately 90% of all UM develop in the choroid, 7% in the ciliary body and 3% in the iris (Damato 2001). UM is the most common primary intraocular malignant tumor (Strickland & Lee 1981) with an annual incidence, which has remained stable for the last 50 years, of 8.4–11.7 cases per million in whites (Bergman et al. 2002). Uveal and Cutaneous Melanoma: Similarities and Differences The incidence of UM in whites is eight times that of Blacks and three times that of Asians (Egan et al. 1988). That fact could point to the casual role of ultraviolet (UV) light as a risk factor for UM (Holly et al. 1990). On the other hand, the incidence of UM in contrast to cutaneous melanoma (CM), has not increased during the last 40 years (Bergman et al. 2002) possibly reflecting a greater role for UV light exposure in the development of CM than in UM (Dolin et al. 1994). Patients with a diagnosis of CM have a 10-fold increased risk of developing a second cutaneous lesion, but they have no additional risk for the development of a UM (Shors et al. 2002). The controversial role of UV exposure in causing UM is only one of a series of differences between the UM and CM. Both melanoma subtypes originate from a common precursor cell, the melanocyte, which migrates from the neural crest to the respective site during the embryonic development period (Soufir et al. 2000). UM metastasizes hematogenously, with the liver frequently being affected (Didolkar et al. 1980) whereas CM tends to spread through the lymphatic system, usually first affecting the regional lymph nodes (Chang et al. 1998). In addition, UM sometimes exclusively spreads to the liver, whereas single organ metastasis is extremely uncommon in CM (Eskelin et al. 2003). Despite the abundant evidence that UM and CM are distinctly separate tumors, few genotypic differences between these melanoma subtypes have been identified. Functional loss of the CDKN2A (p16INK4) tumor suppressor, whether through promoter methylation, mutation, and/or deletion, is observed in a significant fraction of both UMs and CMs (Cannon-Albright et al. 1992; Edmunds et al. 2002; Merbs & Sidransky 1999; van der Velden et al. 2001). Likewise, p53 alterations are observed in both melanoma types in association with tumor progression (Tobal et al. 1992; Weiss et al. 1995). Prognostic Factors of Uveal Melanoma The prognostic factors can be divided in clinical, histopathological, cytogenetical, and molecular. One of the most important clinical prognostic parameters is tumor location where the ciliary body melanoma has a worse prognosis (Seddon et al. 1983) while iris melanomas have the lowest mortality of about 3–5% in 10 years. (Rones & Zimmerman 1958; Shields et al. 2001). Tumor size is another important clinical prognostic factor since high mortality rate is associated with large tumors (Diener-West et al. 1992; COMS 1997). Also tumor configuration is another clinical predictive parameter, where diffuse choroidal melanomas have a poor prognosis (Shields et al. 1996). Cell type, mitotic activity, microcirculation architecture and tumor-infiltrating lymphocytes are the most significant histopathological prognostic factors. Specifically, epithelioid cell type (McLean et al. 1983), high mitotic activity (McLean et al. 1977), presence of microcirculation architecture (vascular patterns) (Rummelt et al. 1995; Folberg et al. 1993) and presence of tumor-infiltrating lymphocytes (de la Cruz et al. 1990) are correlated with poor prognosis. The findings of monosomy 3 and duplication of 8q are associated with a survival rate of 40% at 3 years compared with 95% survival in the absence of these cytogenetic changes. These chromosomal aberrations constitute the most important cytogenetical prognostic factors. (White et al. 1998). The overexpression of c-Myc, a potent inducer of malignant transformation, is known to promote cell growth and proliferation has been unexpectedly associated with improved prognosis (Chana et al. 1998) while the nearby genes DDEF1 and NBS1 were more highly overexpressed and more strongly associated with poor prognostic features (Ehlers & Harbour 2005; Ehlers et al. 2005). Lastly, alterations in tumor-suppressor pathways, such as p53 pathway and the Rb pathway (Janssen et al. 1996; Brantley & Harbour 2000) can play a role in UM. Treatment and Prognosis Treatment modalities of UM include: plaque radiotherapy (brachytherapy) usually with either 106Ru or 125I, (Shields et al. 2000), proton beam radiotherapy (Gragoudas & Marie 2005), transpupillary thermotherapy (Journee-de Korver et al. 2005), trans-scleral local resection (Damato & Foulds 2006), and enucleation (Zimmerman et al. 1978). Systemic disease only rarely responds to treatment and is invariably fatal, usually within 6–9 months of the onset of symptoms. Survival can be much longer (i.e., >1 year) when asymptomatic metastases are detected by screening. UM is the cause of death in approximately 50% of all patients with this disease (Kujala et al. 2003). Age-related Macular Degeneration History, epidemiology, types Since 1874, when it was first described in the medical literature as ‘symmetrical central choroido-retinal disease occurring in senile persons,’ (Hutchinson & Tay 1874) age-related macular degeneration (AMD) has also been referred to as senile, or disciform, macular degeneration, among many other terms. About 25 years ago, the term ‘age-related maculopathy’ was coined and its end stage was acknowledged as AMD. (1) [ Past, current and possible future treatments of age-related macular degeneration. ] Age-related macular degeneration is the leading cause of blindness in individuals aged 55 years and older in industrialized countries (Klein et al. 1995; Dimitrov et al. 2003; Buch et al. 2004). Approximately 1.7% of individuals aged 55 years and older are affected with AMD in Europe (Vingerling et al. 1995) while in the USA an estimated 8 million people are affected from AMD. Age-related Eye Disease Study Research Group (AREDS 2003) Two million people suffer from the advanced form of disease and with the expected increase of people over the age of 85 years, this number will increase to 3 million by the year 2020 (Eye Diseases Prevalence Research Group. 2004; Thylefors 1998). AMD is clinically divided into two forms; the dry or nonneovascular (nonexudative) form comprising about 90% of cases. It is characterized by soft indistinct drusen (63 μm), hyperpigmentation, or depigmentation, and geographic atrophy. Only 10% of patients will develop the wet or neovascular (exudative) form of AMD, which is responsible for nearly 90% of those with severe vision loss. Features of neovascular AMD include subretinal fluid, retinal swelling, hemorrhage, exudation, detachment of the retinal pigment epithelium (RPE) and the development of choroidal neovascularisation (CNV) in addition to those described for nonneovascular AMD (Smith et al. 2007). Pathogenesis, treatment The etiology of AMD is multifactorial and it is believed that four main processes are involved: the accumulation of lipofuscin granules composed mainly of lipids and proteins, as a result of an age-dependent phagocytic and metabolic insufficiency of RPE cells, the accumulation of drusen, amorphous deposits between RPE and the inner collagenous zone of the Bruch′s membrane, the complement factor H (CFH) in the complement cascade which is believed to play an important role in AMD development. (Klein et al. 2005; Donoso et al. 2006). Last but certainly not least, choroidal neovascularization, the proliferation of blood vessels into the subretinal space from the underlying choriocapillaries, is a very complex phenomenon where molecules known as growth factors, among others, regulate angiogenesis (vessel formation). Vascular endothelial growth factor (VEGF) has been identified as a major angiogenic stimulus in the development of CNV (Witmer et al. 2003) but a many other growth factors such as transforming growth factor-β (TGF-ß) (Kliffen et al. 1997) and insulin-like growth factor 1 (IGF-1) (Lambooij et al. 2003) and angiogenic molecules such as angiopoietin (Hera et al. 2005) are believed to play an important role as well. Vitamin and mineral supplementation can reduce the risk of moderate visual loss among some patients with the dry form of AMD as shown from the AREDS (AREDS 2001). For many years laser photocoagulation remained the only available treatment option, although a destructive one, for patients with well-defined extrafoveal CNVs (Moisseiev et al. 1995). Photodynamic therapy with verteporfin, was developed as an alternative to thermal laser photocoagulation could safely reduce the risk of vision loss in patients with subfoveal CNV caused by AMD (TAP 1999). Pegaptanib sodium, an aptamer that binds to VEGF isoform 165 was the first drug to target VEGF and to get Food and Drug Administration (FDA) approval for use against wet AMD. Pegaptanib showed some efficacy in stopping the development of CNV by inhibiting angiogenesis and/or reducing vascular permeability (Gragoudas et al. 2004). Ranibizumab is a humanized anti-VEGF-A recombinant Fab fragment that has been affinity-matured to increase its binding affinity for VEGF-A. Two randomized, double-masked, pivotal phase III clinical trials have demonstrated that monthly intravitreal injection of ranibizumab is effective treatment for subfoveal CNVs in AMD patients, resulting in FDA approval last year (Rosenfeld et al. 2006; Brown et al. 2006). Also bevacizumab, a glycosylated, humanized Fab fragment, approved for metastatic colon cancer and non-small cell lung cancer has showed similar efficacy as ranibizumab in different retrospective studies (Avery et al. 2006; Spaide et al. 2006) and one prospective non-randomized study (Costa et al. 2006). The IGF-1R Structure and function The type 1 insulin-like growth factor receptor (IGF-1R) is a tyrosine kinase (TK) receptor with a 70% homology to the insulin receptor (IR). It was originally considered a redundant receptor used by cells only when signalling from the IR was absent or defective. Both IGF-1R and IR are preformed dimeric TK receptors made up by two extracellular-subunits and two-subunits involving a small extracellular domain, an intramembraneous one as well as an intracellular domain (Adams et al. 2000). The latter includes the juxtamembraneous domain, the TK domain and the C-terminal domain. The IGF-1R and IR are highly homologous, especially in the TK domain in which they share 84% amino-acid identities. However, despite these similarities, the function between IGF-1R and IR differs considerably. (2) [ Simplified scheme of the signalling pathway of the insulin-like growth factor receptor. ] Upon ligand binding, a conformational change induces activation of the kinase. Several docking proteins, such as Src homology 2 domain-containing (Shc) protein and insulin receptor substrates (IRS1-4), are subsequently recruited to the phosphorylation sites in the cytoplasmic domain. Then the signal is propagated via the phosphatidylinositol-3-kinase (PI 3-kinase)/Akt and mitogen-activated protein kinase pathways resulting in cell proliferation and inhibition of apoptosis. IGF-1R signalling can also induce differentiation, malignant transformation and regulate cell–cell adhesion. A dynamic downstream signalling network of different phosphorylation sites of the receptor and cell-context specific recruitment and activation of signalling molecules regulates these different functions (Baserga & Morrione 1999). Furthermore, the IGF-1R can interact with steroid hormones and their receptors, other peptide growth factor receptors, extracellular matrix proteins, integrin receptors and cytokines, such as transforming growth factor-β (Jones & Clemmons 1995). In normal physiology, IGF-1R stimulates linear body growth, promotes neuronal survival and myelination, postnatal mammary development and lactation, and is implicated in bone formation and renal function (Jones & Clemmons 1995). The IGF-1R plays a central role in integrating signals of nutrition and stress into energy shifts from energy-expensive anabolic processes, such as growth and reproduction, to preserving responses under catabolic circumstances (Niedernhofer et al. 2006). The role of IGF-1R in cancer The involvement of the IGF-1R in malignant transformation was first recognised in fibroblasts derived from homozygous IGF-1R null mice embryos (Sell et al. 1993). Mouse embryo fibroblasts are prone to transformation; however, in the absence of IGF-1R they become resistant to malignant transformation by a number of oncogenes [e.g., simian virus 40T antigen (SV40 T) Ewing sarcoma fusion protein]. Re-expression of the IGF-1R restored susceptibility to transformation in these cells. In population studies, high serum levels of IGF-1 have been associated with an increased risk on prostate cancer and premenopausal breast cancer (Renehan et al. 2004). Increased incidence of colorectal adenomas and cancer is seen in acromegaly, in which hypersecretion of GH is accompanied by elevated IGF-1 levels (Jenkins et al. 2006). In vivo overexpression of IGF-1R accelerated the development of tumors in a mouse model of cancer (RIP1-TAG2) (Lopez & Hanahan 2002). In this mouse model, expression of oncoprotein SV40 T in pancreatic islet β cells leads to tumor formation, which is characteristically accompanied by IGF-2 upregulation, providing an activating ligand for the IGF-1R. Remarkably, IGF-1R overexpressing RIP1-TAG2 mice developed more invasive tumors with an increased amount of distant metastases than parental mice. Recently, transgenic expression of a constitutively active IGF-1R fusion protein in mice resulted in spontaneous development of invasive adenocarcinomas of salivary and mammary glands (Carboni et al. 2005). The above-mentioned studies provide proof of the principle that active IGF-1R signalling facilitates malignant transformation, drives growth and progression of established tumors and enhances capability to invade and metastasize. The tumor promoting functions of IGF-1R are embedded in the multi-dimensional process of cancer development and progression. Increased IGF-1R activation, by GH/IGF-1 status or other mechanisms, might create an anti-apoptotic environment thereby favoring cell survival and malignant transformation. Oncogenes, such as the hepatitis B virus oncoprotein or Ewing sarcoma fusion proteins, recruit and activate the IGF-1R signalling pathway by increasing transcription of the IGF-1R gene, while loss of tumor suppressor genes, such as p53, BRCA1 or WT1, results in IGF-1R overexpression by loss of transcriptional control. Overexpression of IGF-1R in tumor compared to normal tissue is shown in a number of studies with polymerase chain reaction (PCR) detection of IGF-1R mRNA (Werner et al. 2000; Weber et al. 2002). Amplification of the IGF-1R gene, however, is infrequent, as shown in breast tumors (<2%) (Almeida et al. 1994) and (Berns et al. 1992) and sarcomas (Sekyi-Out et al. 1995). Activating mutations of the receptor have not been described yet. Numerous other molecular mechanisms that modulate IGF-1R signalling in cancer, such as loss of imprinting of IGF-2, altered glycosylation and constitutive activation of downstream proteins, have been described (Samani et al. 2007). Up-regulation of IGF-1R signalling has recently been implicated in the development of resistance to anti-cancer therapy, such as radiotherapy, hormonal therapy and human epidermal growth factor receptor 2 targeting (Nahta et al. 2006; Milano et al. 2006). Targeting IGF-1R in cancer The vast expression of IGF-1R in neoplastic cells and tissues combined with its crucial roles in cancer cell growth makes this tyrosine receptor an attractive target to combat malignant diseases. A variety of approaches aimed at targeting IGF-1R has been utilised to prove the concept, or are being developed as potential anticancer therapies. Targeting IGF-1R to block its signalling may be obtained by interference with ligand/receptor interactions, receptor synthesis and expression, receptor TK activity, or combinations of these strategies. Strategies aimed to block the ligand–receptor interaction involve receptor neutralizing antibodies (Kalebic et al. 1994). Among those most studied is the monoclonal antibody-IR3, which competes with IGF-1 for binding to the receptor and blocks receptor activation (Van Wyk et al. 1985). Antibody blockade of IGF-1R has been attempted in breast cancer model systems. However, the large size of the therapeutic molecule restricts its access to tumor cells, particularly in the central regions of solid tumors (Russell et al. 1992). Smaller fragments are currently being studied as a substitute for whole antibodies in an effort to improve access and uptake. Sachdev et al. (2003) used a single-chain antibody directed against IGF-1R (IGF-1R scFv-Fc) to examine the effects on IGF-1R signalling. In vivo treatment of mice bearing MCF-7 xenograft tumors with scFv-Fc resulted in near complete downregulation of IGF-1R. Antisense techniques are another way to inactivate the IGF-1R. Resnicoff et al. (1994) used antisense RNA to IGF-1R by introducing it into cells by either addition of oligodeoxynucleotides or by transfection with plasmids expressing antisense RNA to IGF-1R RNA. Injection of glioblastoma cells (C6) IGF-1R antisense cells into rats carrying an established wild-type C6 tumor caused complete regression of the tumors. This indicate that practical applications may be developed to target IGF-1R. A direct strategy to interfere with IGF-1R activity is to induce selective inhibition of its TK by developing selective small-molecular inhibitors. The major advantage of this approach is that small molecules have a considerable higher bioavailability compared to antibodies, dominant-negative receptors and antisense oligonucleotides. However, TK inhibitors face the problem that IGF-1R and IR are so similar. Actually many of the hitherto developed IGF-1R TK inhibitors have also caused substantial inhibition of the IR. Such cross reaction would probably cause diabetic reactions in patients and can therefore not be accepted. On the other hand, IR-A dependent tumors would not be affected by a fully selective IGF-1R inhibitor. Most of the IGF-1R TK inhibitors produced so far have served as competitive ATP inhibitors. Since the region of the TK domain covering the ATP binding site is identical to that of the IR, such cross-inhibitions are not unexpected. However, there is a recent interesting exception. Garcia-Echeverria et al. (2004) presented a new compound (a pyrrolo[2,3-d] pyrimidine) that although inhibiting the IGF-1R and IR TK equipotently in cell-free systems, exhibited several-fold selectivity for the IGF-1R in a cellular context and reduced the growth of IGF-1R positive fibrosarcomas in vivo. Blum et al. (2003) presented a new family of bioisostere inhibitors, based on the structure of AG 538, a tyrphostin inhibiting the IGF-1R TK at the substrate level and not at the ATP binding site (Blum et al. 2000). These AG 538 bioisosteres possessed similar but weaker biological properties to AG 538 but are more stable and blocked the formation of colonies of prostate and breast cancer cells in soft agar systems (Blum et al. 2003). Recently, we demonstrated that the cyclolignan PPP inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity (Girnita et al. 2004), as well as it reduced phosphorylated Akt (Girnita et al. 2004; Girnita et al. 2005; Vasilcanu et al. 2004; Colon et al. 2007) caused apoptosis and induced tumor regression in xenografted mice. PPP did not compete with ATP but interfered with phosphorylation in the activation loop of the kinase domain, in which it blocked phosphorylation of the tyrosine (Y1136) residue, while sparing the two others (Y1131 and Y1135). Since an IGF-1R construct, in which the tyrosine at position 1136 was replaced by a phenylalanine, also led to a strong inhibition of phosphorylated Akt in transfected cells, it was suggested that this mechanism may be responsible for the apoptotic effect of PPP (Vasilcanu et al. 2004). The IGF-1R has been shown to play a central role in transformation and tumorigenesis. Targeting of IGF-1R signalling by many different approaches has caused a reversal of the transformed phenotype and has induced apoptosis in vitro and in vivo. Moreover, cancer cells are sensitized to conventional chemotherapeutic treatment and irradiation. Therefore, the IGF-1R is a promising target for a specific and selective tumor therapy. Aims of the Study • To determine whether c-Met and IGF-1R could be used as prognostic markers for UM using immunohistochemistry. • To investigate whether targeting of IGF-1R with an inhibitor like PPP, could affect the metastatic meccanisms of UM. • To evaluate the efficacy of oral PPP in UM in vivo and look for synergism of PPP with different anti-tumor agents. • To investigate the effect of PPP on a animal CNV model and its effect on VEGF secretion from RPE cells. Materials and Methods Clinical material Primary UM specimens, fixed in formaldehyde and paraffin embedded, from 152 consecutive patients that undergone enucleation were available for this study. Twenty lesions were deemed extensively necrotic (defined as >50% of cells necrotic) and excluded from further evaluation, leaving 132 lesions to be immunostained as outlined herein. These lesions were from 55 female and 77 male patients (average age, 63 years; range, 25–85). Antibodies In paper I, a rabbit polyclonal antibody directed to the human IGF-1R (N-20) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and a mouse monoclonal antibody directed to human c-Met (NCL-cMET) was provided by Immunkemi (Novocastra Ltd., Newcastle-upon-Tyne, UK). In paper II, III and IV the phosphotyrosine (PY99) and polyclonal antibodies to the β-subunit of IGF-1R (H-60) were from Santa Cruz Biotechnology Inc., while in paper IV polyclonal antibodies to to IGF-1(H-70), to VEGF (VG-1) and to GAPDH (FL-335) were from Santa-Cruz Biotechnology Inc. Reagents PPP was synthesized as described (Girnita et al. 2004) and was dissolved in DMSO (0.5 mmol/l) before addition to cell cultures. PPP/food mixture was prepared as follows: One hundred and eighty mg of PPP was first dissolved in 50 ml acetone. The solution was then added to a mixture of 500 mg of food powder (Lactamin, Kimstad, Sweden) and acetone: sterile water (1:1) to make the food semi-liquid. The final solution was then mixed thoroughly with a mixer and dried overnight at 40°. Imatinib-mesylate (Gleevec) was a gift from Novartis Pharmaceuticals (Basel, Switzerland). Cisplatin, doxorubicin and 5-FU was a kind gift from Professor Linder (CCK, Karolinska Institute, Stockholm). Cell cultures Four cell lines obtained from human primary UMs (OCM-1, OCM-3, OCM-8 and 92-1) were previously described (All-Ericsson et al. 2002). R+ cells (overexpressing the human IGF-IR) were from Dr Renato Baserga (Thomas Jefferson University, Philadelphia, PA, USA). R+ cell line was cultured in the presence of G-418 (Promega, Madison, WI, USA). R-v-src were from Dr Renato Baserga (Thomas Jefferson University). The R-v-src fibroblasts are R-cells transfected with the v-src (being the only single oncogene that can bypass the requirement for a functional IGF-1 receptor in anchorage-independent growth), and have IR substrate-1 and Shc constitutively tyrosine phosphorylated. ARPE-19 is a non-transformed human diploid RPE cell line that displays many differentiated properties typical of RPE in vivo. (Dunn et al. 1996) ARPE-19 cells were plated at subconfluency and maintained in culture at 37° in 5% CO2. All ARPE-19 cultures were maintained at 37° in 5% CO2 in Dulbecco’s modified Eagle’s medium: nutrient mixture F12 plus 10% fetal bovine serum. Immunohistochemistry In paper I immunostaining of tissue sections was performed by using the standard avidin–biotin complex (ABC) technique (Vector Laboratories, Burlingame, CA, USA). Briefly, 4-μm tissue sections were cut from each of the selected 132 paraffin-embedded tumor specimens. Tissue sections were deparaffinized and rehydrated. The samples were bleached with hydrogen peroxide-disodiumhydrogenphosphate at room temperature – IGF-1R samples overnight and c-Met 3 hr only, because they were subjected to antigen retrieval according to the manufacturer’s instructions. After antigen retrieval (c-Met) tissue sections were rinsed in Tris-buffered saline (TBS, pH 7.6) and incubated with blocking serum (1% bovine serum albumin) for 20 min at room temperature followed by an overnight incubation at 8° with an excess of anti c-Met antibody or anti-IGF-1R antibody. Biotinylated anti-mouse monoclonal IgG and anti-rabbit polyclonal IgG antibodies were added for c-Met and IGF-1R, respectively, and incubation was continued for an additional 30 min at room temperature, followed by application of the ABC complex. The peroxidase reaction was developed for 6 min at room temperature with 0.6 mg/ml 3′3-diaminobenzidine tetrahydrochloride with 0.03% hydrogen peroxide. Counterstaining was performed with Mayer’s hematoxylin. TBS was used for rinsing between the different steps. Appropriate positive and negative controls were included. Staining assessment In paper I, all stained cells were considered positive, irrespective of staining intensity. We scored the results of c-Met and IGF-1R immunoexpression as negative when no staining was present, low when <10% of cells were stained, moderate when 10–50% of cells were stained, and high when more than 50% of cells were immunoreactive. At a later stage, and without knowledge of the initial result, the same observer (ME) repeated the assessment for a random sample of slides from 30 UM specimens. These slides were also independently assessed by an experienced ophthalmic pathologist (SS) using the same grading system. The interobserver reproducibility according to the test was 0.73 (95% CI, 0.54–0.92), and the intraobserver reproducibility was 0.69 (95% CI, 0.49–0.89). Both observers were masked to results from earlier assessments and to survival data. Immunoprecipitation and Western blotting In paper II, cells were cultured to subconfluency in 6-cm plates. After one day the cells were serum-depleted for 24 hr before addition of PPP, and then stimulated by IGF1 for 5 min. For determination of IGF1 phosphorylation, the cells were then lyzed and subjected to immunoprecipitation by adding 20 μl of resuspended volume of the sepharose conjugate (Protein G-Sepharose) and incubation at 4° with the anti-IGF-1R β antibody H-60. Immunoprecipitants were analyzed by Western blotting. Antibodies to IGF-1R β-unit were used as loading control. In paper III, tumor samples obtained from the in vivo experiments with OCM-1 and OCM-8 cells were analyzed. Samples from fresh-frozen tumors from drug- and solvent-treated mice were cut in pieces and suspended in freshly prepared homogenization buffer as described (Girnita et al. 2004). After centrifugation at 14 000 × g for 10 min at 4° the supernatants were immunoprecipitated for IGF-1R and IR, and determination of IGF-1R and IR phosphorylation, after indicated treatments, was completed. Fifteen microlitre Protein G Plus-A/G agarose and 1μg antibody were added to 1 mg of protein material. After overnight incubation at 4° on a rocker platform, the immunoprecipitates were collected by centrifugation in a microcentrifuge at 2500 rpm (∼5000 × g) for 2 min. The supernatant was discarded, whereupon the pellet was washed and then dissolved in a sample buffer for SDS-PAGE. Twenty micrograms of protein per sample were electrophoresed in a 10% Tris–Glycin gel (Novex, Invitrogen, Carlsbad, CA, USA). Following electrophoresis the proteins were transferred overnight to nitrocellulose membranes (Amersham, Uppsala, Sweden) and then blocked for 1 hr at room temperature in a solution of 5% (w/v) skimmed milk powder and 0.02% (w/v) Tween 20 in PBS, pH 7.5. Incubation with appropriate primary antibodies for IGF-1R, IR and VEGF was performed for 1 hr at room temperature, or overnight at 4°. This was followed by washes with PBS and incubation with either a HRP-labeled or a biotinylated secondary antibody (" @default.
• W4211072571 created "2022-02-13" @default.
• W4211072571 date "2008-11-19" @default.
• W4211072571 modified "2023-10-17" @default.
• W4211072571 title "Introduction: Uveal melanoma" @default.
• W4211072571 cites W109974915 @default.
• W4211072571 cites W123314342 @default.
• W4211072571 cites W1487528435 @default.
• W4211072571 cites W1545177819 @default.
• W4211072571 cites W1562028693 @default.
• W4211072571 cites W1588612022 @default.
• W4211072571 cites W1601975653 @default.
• W4211072571 cites W1802218256 @default.
• W4211072571 cites W1965110077 @default.
• W4211072571 cites W1967790688 @default.
• W4211072571 cites W1969328947 @default.
• W4211072571 cites W1972486056 @default.
• W4211072571 cites W1976305765 @default.
• W4211072571 cites W1977158511 @default.
• W4211072571 cites W1981148605 @default.
• W4211072571 cites W1982314128 @default.
• W4211072571 cites W1983433202 @default.
• W4211072571 cites W1986145457 @default.
• W4211072571 cites W1987890351 @default.
• W4211072571 cites W1998217029 @default.
• W4211072571 cites W1999030960 @default.
• W4211072571 cites W1999575468 @default.
• W4211072571 cites W1999611557 @default.
• W4211072571 cites W2000292756 @default.
• W4211072571 cites W2003133121 @default.
• W4211072571 cites W2003776674 @default.
• W4211072571 cites W2007852946 @default.
• W4211072571 cites W2009218955 @default.
• W4211072571 cites W2009876678 @default.
• W4211072571 cites W2010296481 @default.
• W4211072571 cites W2011133384 @default.
• W4211072571 cites W2012940389 @default.
• W4211072571 cites W2014262167 @default.
• W4211072571 cites W2017613801 @default.
• W4211072571 cites W2018503840 @default.
• W4211072571 cites W2021159608 @default.
• W4211072571 cites W2021942056 @default.
• W4211072571 cites W2022544748 @default.
• W4211072571 cites W2025432795 @default.
• W4211072571 cites W2027998941 @default.
• W4211072571 cites W2029482683 @default.
• W4211072571 cites W2030954888 @default.
• W4211072571 cites W2032409176 @default.
• W4211072571 cites W2033245558 @default.
• W4211072571 cites W2033587812 @default.
• W4211072571 cites W2037348124 @default.
• W4211072571 cites W2038198672 @default.
• W4211072571 cites W2039327658 @default.
• W4211072571 cites W2040577381 @default.
• W4211072571 cites W2041653037 @default.
• W4211072571 cites W2046193969 @default.
• W4211072571 cites W2047352791 @default.
• W4211072571 cites W2047393488 @default.
• W4211072571 cites W2051842051 @default.
• W4211072571 cites W2055237105 @default.
• W4211072571 cites W2055835855 @default.
• W4211072571 cites W2060922823 @default.
• W4211072571 cites W2067233601 @default.
• W4211072571 cites W2068944371 @default.
• W4211072571 cites W2071423177 @default.
• W4211072571 cites W2072653487 @default.
• W4211072571 cites W2073786886 @default.
• W4211072571 cites W2078027523 @default.
• W4211072571 cites W2082025128 @default.
• W4211072571 cites W2082501870 @default.
• W4211072571 cites W2085712747 @default.
• W4211072571 cites W2088831705 @default.
• W4211072571 cites W2089223199 @default.
• W4211072571 cites W2091253961 @default.
• W4211072571 cites W2095313197 @default.
• W4211072571 cites W2110920908 @default.
• W4211072571 cites W2111843196 @default.
• W4211072571 cites W2112480698 @default.
• W4211072571 cites W2113784410 @default.
• W4211072571 cites W2114112168 @default.
• W4211072571 cites W2117012110 @default.
• W4211072571 cites W2117133523 @default.
• W4211072571 cites W2117891396 @default.
• W4211072571 cites W2121176463 @default.
• W4211072571 cites W2124415048 @default.
• W4211072571 cites W2125652724 @default.
• W4211072571 cites W2127644935 @default.
• W4211072571 cites W2132503502 @default.
• W4211072571 cites W2145908666 @default.
• W4211072571 cites W2148111934 @default.
• W4211072571 cites W2161708429 @default.
• W4211072571 cites W2165062842 @default.
• W4211072571 cites W2166075836 @default.
• W4211072571 cites W2168912282 @default.
• W4211072571 cites W2169148310 @default.
• W4211072571 cites W2169392527 @default.
• W4211072571 cites W2292658304 @default.
• W4211072571 cites W2396478796 @default.

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