Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

BACKGROUND/AIMS: A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma. METHODS: 90 eyes were enucleated from 89 patients for retinoblastoma (n = 24) or for choroidal or ciliary body melanoma (n = 66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1. RESULTS: Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed. CONCLUSION: Retinoblastomas and uveal melanomas, two tumours of differing pRB status, differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.     

Human hydroxyindole-O-methyltransferase in pineal gland, retina and Y79 retinoblastoma cells

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) was studied in extracts of human pineal gland, retina and Y79 retinoblastoma cells. HIOMT enzyme activity and immunoreactive protein (approximately 42 kDa) were undetectable in the human retina; very low levels of HIOMT mRNA were detected using a highly sensitive RT-PCR/Southern blot method, as has been reported. Analysis of extracts of Y79 cells indicated that HIOMT enzyme activity, immunoreactivity (approximately 42 kDa) and mRNA (approximately 1.3 kb) were detectable at approximately 1/5-1/40 the levels found in the pineal gland. This unambiguously establishes that the HIOMT gene is expressed in Y79 cells. Kinetic analysis of Y79- and pineal-derived HIOMT indicates that the enzyme is generally similar in both tissues; one difference, however, is that substrate inhibition by N-acetylserotonin is greater with the Y79-derived enzyme. These studies show that Y79 cells represent a valid model to study the regulation of human HIOMT protein and mRNA; the differences detected may reflect the existence of tissue-specific regulatory mechanisms or differential patterns of expression of HIOMT isoforms.     

Rubeosis iridis in retinoblastoma. Histologic findings and the possible role of vascular endothelial growth factor in its induction

PURPOSE: Iris neovascularization (rubeosis iridis) is a common finding in eyes harboring retinoblastoma. The purpose of the current study is to evaluate the histologic factors that may affect the development of rubeosis iridis in eyes with retinoblastoma and to examine whether vascular endothelial growth factor (VEGF), a hypoxia-induced angiogenic factor, is produced by hypoxic retinoblastoma and retinal cells in these eyes. MATERIALS AND METHODS: One hundred eighty-one enucleated eyes containing retinoblastoma were the source for the current study. Histologic slides were evaluated for the presence and degree of rubeosis iridis as well as for other histologic factors. Univariate and multivariate statistical analyses were performed to find a correlation between rubeosis iridis and the other histologic factors. Eight of the eyes underwent in situ hybridization with a specific VEGF mRNA probe to locate tumor and retinal cells that may produce this hypoxia-induced angiogenic factor. RESULTS: The amount of tumor necrosis as well as choroidal and optic nerve invasion was found to be one of the most important factors that correlated with the presence and degree of rubeosis iridis in the examined eyes. All eight eyes that underwent in situ hybridization analysis showed strong signals of VEGF mRNA in retinoblastoma cells around necrotic regions and in the outer nuclear layers in areas of detached retina. CONCLUSIONS: There exists an association between rubeosis iridis and histologic factors found in advanced stages of retinoblastoma, especially the amount of tumor necrosis. Vascular endothelial growth factor may well be an angiogenic factor that is secreted by the hypoxic retinoblastoma and retinal cells and, reaching the iris, causes (presumably in collaboration with other factors) rubeosis iridis.     

Enhanced expression of vascular endothelial growth factor in metastatic melanoma

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.     

Putative control of angiogenesis in hemangioblastomas by the von Hippel-Lindau tumor suppressor gene

The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.     

Atmospheric deposition levels of chosen elements in the Czech Republic determined in the framework of the International Bryomonitoring Program 1995

Human prostate specimens commonly contain a spectrum of epithelial changes, including normal acinar and ductal structures, hyperplasia, intraepithelial neoplasia (dysplasia), and carcinoma. Since vascular endothelial growth factor (VEGF) expression is dependent on cell type and tissue microenvironment, meaningful quantitation of the levels of this mRNA in pathological specimens requires analysis at the microscopic level. Phosphorimage analysis of the binding of radiolabeled cRNA probes to tissue sections allows quantitation of mRNA levels, but the resolution is limited. Alternatively, emulsion autoradiography allows visualization of mRNA levels at cellular resolution, but quantitation is difficult. We have developed a method of quantitating steady state mRNA levels in tissue sections at the microscopic level, using autoradiography and quantitative image analysis. In this study, we describe the method and apply it to quantitation of VEGF mRNA in human prostate specimens. The VEGF mRNA level was low in nonepithelial stromal tissue (0.8 dpm/mm2), high in normal and benign hyperplastic epithelium (17-18 dpm/mm2), and significantly decreased in intraepithelial neoplasia (6.4 dpm/mm2) and in microacinar carcinoma that had invaded the stroma (3.5 dpm/mm2). Immunohistochemical staining detected VEGF protein in epithelial and stromal cells, with highest levels on the luminal surface of normal epithelium and in stromal cells, and lower levels in benign hyperplasia, intraepithelial neoplasia, and carcinoma. No correlation between VEGF expression in epithelium and nearby vessel density was observed. The results indicate a decrease in the steady state level of VEGF mRNA when prostate epithelial cells become transformed, escape the confines of glandular structure and invade the stroma, and suggest that the progression of prostatic carcinoma through the stages examined in this study is not associated with increased VEGF expression, in contrast to the elevated VEGF expression associated with progression of several other tumor types.     

Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

"Mother-baby" biliary endoscopy: the University of Chicago experience

Recoverin is a calcium-binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 mM butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite-like cellular processes. Addition of 4 mM dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.     

Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function

Hemangioblastomas are highly vascular tumors of the central nervous system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo expression in hemangioblastomas was observed only in individual cases, and the analyses were mainly based on indirect determination of erythropoiesis-stimulating activity. Therefore, we analyzed a series of 11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot analysis and compared the results with normal brain and glioblastomas. Surprisingly, we observed Epo mRNA expression in all hemangioblastoma specimens analyzed, but in none of four glioblastomas. In contrast, VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas. In situ hybridization revealed neoplastic stromal cells as Epo- and VEGF-producing cells in hemangioblastomas. These results suggest that in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to VEGF, might be negatively regulated by the VHL gene product.     

Uveal melanoma, choroidal hemangioma, and simulating lesions. Role of MR imaging

Malignant uveal melanomas are the most common primary intraocular malignant tumors in adults. Choroidal hemangiomas are congenital, benign, vascular lesions that may be confused with malignant uveal melanomas on clinical examination. The MR imaging characteristics of uveal melanomas and choroidal hemangiomas are different, making diagnostic differentiation possible with a high degree of accuracy. This article describes the MR imaging features of uveal melanoma, choroidal hemangioma, and other simulating lesions.     

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

Expression of vascular endothelial growth factor in normal liver and hepatocellular carcinoma: an immunohistochemical study

Angiogenesis is of vital importance during the development and progression of solid tumors. To examine the role of vascular endothelial growth factor (VEGF) in hepatocarcinogenesis, we evaluated the expression of peptide in normal human liver (n = 6) and in 36 cases of hepatocellular carcinoma (HCC). Immunoreactivity for VEGF was present in the extracellular matrix of the portal tracts in the normal and nontumor part of liver, but not in hepatocytes and bile duct epithelium. For HCC, variable amounts of VEGF were expressed in 13 cases (36.1%) of tumor cells. Using a logistic regression model, expression of VEGF was significantly associated with a higher proliferative index (P = .01) and sonographic portal vein thrombosis (P = .05). However, VEGF expression did not correlate with a biochemical liver profile, alpha-fetoprotein levels, histological grading, gender, or clinical stage of cirrhosis (P > 0.1, respectively). Log-rank test showed that evaluation of VEGF did not provide more prognostic information (P > .5) than that from tumor volume and portal vein thrombosis (P < .01, respectively). In addition, VEGF was always present in the fibrovascular stroma or pericellular matrix of HCC, although no strong relationship was observed with the expression of VEGF in tumor cells (P > .5). Our data suggested that expression of VEGF may characterize a progression toward higher proliferation in hepatocarcinogenesis in vivo. The relevance of VEGF existing in the extracellular matrix of the normal liver and HCC remains to be clarified.     

Recommendations on the contents of medical reports in rectal carcinoma: II: Physician''s letter. Orienting guides in the interdisciplinary patient care process

Vascular endothelial growth factor (VEGF) is a hypoxia inducible angiogenic and vascular permeability factor. Although VEGF expression in glioblastoma is induced by hypoxia, its expression in renal cell carcinoma and hemangioblastoma is thought to be related to mutation of the von Hippel-Lindau (VHL) gene. It is not certain whether other lesions in VHL syndrome are associated with an elevated VEGF level. We report a VHL syndrome patient with multiple hemangioblastomas and bilateral epididymal clear cell papillary cystadenomas. In situ hybridization revealed high levels of VEGF mRNA in the clear cells of the epididymal tumor and the stromal cells of the hemangioblastoma. This lends support to the notion that upregulation of VEGF is caused by loss of the wild-type VHL protein. We postulate that the elevated VEGF levels may account for the cyst formation and vascularized stroma present in these VHL-associated tumors.     

Expenditures for the treatment of major depression

At present, little information is available on specific chromosomal aberrations in malignant melanomas of different subtypes and different growth behaviors. Therefore, we have applied fluorescence in situ hybridization on isolated interphase cells from paraffin sections of 79 primary tumors of malignant melanomas: 47 nodular melanomas and 32 superficial spreading melanomas in various stages. We used centromeric probes for the chromosomes 1, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X, and Y and a midisatellite probe localized in 1p36. The number of chromosomal aberrations and the ploidy of the cells rose with the tumor stage in both subtypes, although in superficial spreading melanomas, fewer chromosomal abnormalities were detectable than in nodular melanomas. A deletion in 1p36 could only be found in nodular melanomas (mostly in higher tumor stages), not in superficial spreading melanomas. Our results show that the different histologic subtypes of malignant melanoma of the skin differ also in their chromosomal aberrations. In addition, it seems that there may be a correlation between the growth characteristics and putative tumor suppressor genes on 1p36.     

Human cytomegalovirus infection in a retinoblastoma cell line in vitro

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.     

Involvement of interleukin-1beta-converting enzyme in apoptosis of irradiated retinoblastomas

PURPOSE: To investigate whether interleukin-1beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, is involved in gamma-irradiation-induced apoptosis (programmed cell death) of human retinoblastoma cells. METHODS: The induction of apoptotic cell death in human retinoblastoma cell lines WERI-Rb-1 and Y79 by gamma-irradiation was determined with a modified 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide colorimetric assay and the DNA-binding fluorochrome bis (benzimide) trihydro-chloride (Hoechst 33258) staining. The change of ICE protein level in tumor cells during apoptosis was determined by immunoblotting assay. Whether the specific tetrapeptide ICE inhibitor Ac-YVAD-CMK affected gamma-irradiation-induced apoptosis in tumor cells was also examined. The effect of ICE overexpression on tumor cells was evaluated by a transient transfection assay using ICE expression vector. RESULTS: Gamma-irradiation inhibited the cell viability of WERI-Rb-1 and Y79 cells in a dose-dependent manner and induced apoptosis. The protein level of ICE was remarkably enhanced after the treatment. The apoptotic cell death induced by gamma-irradiation was suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK. Moreover, overexpression of ICE induced apoptosis in tumor cells. CONCLUSIONS: These findings suggest that ICE may play an important role in gamma-irradiation-induced apoptosis in retinoblastoma cells. Transfer of the ICE gene induces apoptosis in these cells without gamma-irradiation.     

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.     

Establishment and characterization of human ocular melanoma cell lines

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.