RESUMEN

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

Asunto(s)

Anomalías Múltiples/genética , Blefaroptosis/congénito , Duplicación Cromosómica , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Adulto , Animales , Blefaroptosis/genética , Estatura/genética , Niño , Fisura del Paladar/genética , Femenino , Dedos/anomalías , Humanos , Discapacidad Intelectual/genética , Cariotipificación , Masculino , Ratones , Ratones Transgénicos , Microcefalia/genética , Síndrome

RESUMEN

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and beta-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-beta increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

Asunto(s)

Proteoglicanos de Heparán Sulfato/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Animales/metabolismo , Animales , Northern Blotting , Western Blotting , Cadherinas/metabolismo , Línea Celular Tumoral , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Femenino , Glipicanos , Proteoglicanos de Heparán Sulfato/metabolismo , Inmunohistoquímica , Factor II del Crecimiento Similar a la Insulina/farmacología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Metástasis de la Neoplasia , Ratas , Transactivadores/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacología , beta Catenina

RESUMEN

Glypican-3 (GPC3) is a membrane-bound heparan sulfate proteoglycan that is mutated in the Simpson-Golabi-Behmel syndrome. This is an X-linked condition characterized by overgrowth, and various visceral and skeletal dysmorphisms. The phenotype of the Simpson-Golabi-Behmel syndrome patients and GPC3-deficient mice, as well as gene transfection experiments indicate that GPC3 can act as an inhibitor of cell proliferation and survival. It has been previously shown that GPC3 expression is downregulated in mesotheliomas and ovarian cancer. Here we report that GPC3 expression is also silenced in human breast cancer, and that this silencing is due, at least in part, to hypermethylation of the GPC3 promoter. Ectopic expression of GPC3 inhibited growth in eight out of 10 breast cancer cell lines. Collectively, these data suggest that GPC3 can act as a negative regulator of breast cancer growth.

Asunto(s)

Azacitidina/análogos & derivados , Neoplasias de la Mama/genética , Metilación de ADN , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Proteoglicanos de Heparán Sulfato/genética , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Azacitidina/farmacología , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/química , Decitabina , Compensación de Dosificación (Genética) , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glipicanos , Proteoglicanos de Heparán Sulfato/biosíntesis , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Pérdida de Heterocigocidad , Proteínas de Neoplasias/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Cromosoma X/genética

RESUMEN

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor-alpha (TGF-alpha), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF-alpha exerts its anti-anoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-alpha. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X(L), another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Src-dependent manner. We conclude that TGF-alpha has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X(L) expression induced by detachment from the ECM.

Asunto(s)

Apoptosis , Mucosa Intestinal/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Animales , Proteína Tirosina Quinasa CSK , Caspasa 3 , Inhibidores de Caspasas , Caspasas/biosíntesis , Células Cultivadas , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Mucosa Intestinal/citología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Ratas , Proteína bcl-X , Familia-src Quinasas

Asunto(s)

Proteínas de Drosophila , Proteoglicanos de Heparán Sulfato/fisiología , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Animales , Constitución Corporal/genética , Progresión de la Enfermedad , Drosophila melanogaster/genética , Femenino , Genes de Insecto , Gigantismo/genética , Gigantismo/metabolismo , Glicosilfosfatidilinositoles/química , Glipicanos , Sustancias de Crecimiento/metabolismo , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/clasificación , Proteoglicanos de Heparán Sulfato/genética , Humanos , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Conformación Proteica , Proteoglicanos/genética , Proteoglicanos/fisiología , Ratas , Síndrome , Cromosoma X/genética

RESUMEN

Normal or immortal epithelial cells are sensitive to a form of apoptosis, commonly referred to as anoikis, which is induced by detachment from the extracellular matrix (ECM). In contrast, development of carcinomas is associated with acquisition of cellular resistance to anoikis. However, whether human cancer cells deprived of anoikis resistance necessarily display reduced tumorigenic properties in vivo is unknown. We decided to address this question using human ovarian carcinoma cells as a model. Bcl-X(L), an apoptotic factor considered to play an important role in (resistance to) anoikis, is overexpressed in ovarian cancer, and represents an unfavorable prognostic indicator for this type of human malignancy. We therefore evaluated whether Bcl-X(L) can be used as a tool to manipulate anoikis resistance and tumorigenicity of ovarian cancer cells. We show here that when nonmalignant ovarian epithelial cells are detached from the ECM, down-regulation of Bcl-X(L) and apoptotic cell death are observed, although these events do not occur in ovarian carcinoma cells. Moreover, enforced down-regulation of Bcl-X(L) by transfection with antisense cDNA in the anoikis-resistant and highly tumorigenic HEY ovarian carcinoma cell line had no impact on the viability of these cells under adherent conditions but caused significant apoptosis in response to detachment from the ECM. This change was associated with a strong inhibition of tumorigenicity of the Bcl-X(L)-deficient HEY cells in nude mice, both s.c. and in the peritoneal cavity. These results suggest a critical role for Bcl-X(L) in the maintenance of anoikis resistance in ovarian cancer cells. They also serve to establish a functional linkage between this property and the ability of human cancer cells to grow aggressively in vivo. Consequently, targeting molecular mechanisms responsible for anoikis resistance may serve as a potentially effective therapeutic strategy for the treatment of such human malignancies as ovarian cancer.

Asunto(s)

Anoicis/fisiología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , División Celular/fisiología , ADN sin Sentido/genética , ADN Complementario/genética , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Esferoides Celulares/patología , Transfección , Proteína bcl-X

RESUMEN

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.

Asunto(s)

Apoptosis , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Grupo Citocromo c/metabolismo , Ratas , Proteína X Asociada a bcl-2

RESUMEN

The name glypican has been assigned to a family of heparan sulfate (HS) proteoglycans that are linked to the cell membrane by a glycosyl-phosphatidylinositol anchor. To date, six family members of this family have been identified in mammals (GPC1 to GPC6) and two in Drosophila. Glypicans are expressed predominantly during development, and they are thought to play a role in morphogenesis. As HS-carrying molecules, glypicans were initially considered potential regulators of heparin-binding growth factors. This has been recently confirmed by genetic interaction experiments showing that glypicans regulate wingless signaling in Drosophila. The involvement of glypicans in the in vivo regulation of other heparin-binding growth factors, such as fibroblast growth factors, remains to be determined. Interestingly and unexpectedly, a role for GPC3 in the regulation of insulin-like growth factors has been proposed. This hypothesis is based on the phenotype of patients with Simpson-Golabi-Behmel syndrome (SGBS), an overgrowth and dysmorphic syndrome in which the GPC3 gene is mutated. Thus, it is possible that glypicans regulate different kinds of growth factors in a tissue-specific manner. In addition to its involvement in SGBS, down-regulation of GPC3 has been recently associated with the progression of several types of malignant tumors, including mesotheliomas and ovarian cancer. A role for GPC1 in pancreatic cancer progression has also been proposed.

Asunto(s)

División Celular/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Neoplasias/patología , Animales , Ratones

RESUMEN

The kidney of the Gpc3-/ mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Here, we identify cellular and molecular mechanisms underlying this renal dysplasia. Glypican-3 (GPC3) deficiency was associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation was increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggested that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogated the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converted BMP7-dependent inhibition to stimulation, and enhanced the stimulatory effects of KGF. Similar comparative differences were found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. We propose that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis.

Asunto(s)

Proteínas Morfogenéticas Óseas/farmacología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Proteoglicanos de Heparán Sulfato/fisiología , Riñón/embriología , Animales , Apoptosis , División Celular , Femenino , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Glipicanos , Ratones , Ratones Endogámicos C57BL , Morfogénesis

RESUMEN

Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-X(L), and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-X(L) in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-X(L) is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-X(L) was PI 3-kinase- and mitogen-activated protein kinase (MAP kinase)-independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-X(L) expression in the absence of the ECM.

Asunto(s)

Apoptosis/fisiología , Transformación Celular Neoplásica , Matriz Extracelular/fisiología , Genes ras , Mucosa Intestinal/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular , Línea Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oligonucleótidos Antisentido/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Transfección , Proteína bcl-X

RESUMEN

A possible link between oncogenes and tumor angiogenesis has been implicated by the finding that expression of various oncogenes, particularly mutant ras, can lead to a marked induction of a potent paracrine stimulator of angiogenesis, vascular endothelial growth factor (VEGF). We sought to determine how oncogenic ras induction of VEGF is mediated at the molecular level and whether the mechanisms involved differ fundamentally between transformed epithelial cells and fibroblasts. Our results suggest that in a subline (called RAS-3) of immortalized nontumorigenic rat intestinal epithelial cells (IEC-18) that acquired a tumorigenic phenotype upon transfection of mutant ras, up-regulation of VEGF occurs in the absence of an autocrine growth factor circuit. The expression of VEGF mRNA and protein by RAS-3 cells was strongly suppressed in the presence of LY294002, an inhibitor of phosphatidylinositol 3'-kinase, but remained largely unaffected in the same cells treated with an inhibitor (PD98059) of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MKK/MEK-1). This is consistent with the observation that overexpression of a constitutively activated mutant of MEK-1 (AN3/ S222D) in the parental IEC-18 cells did not result in up-regulation of VEGF production. The impact of mutant ras on VEGF expression was also significantly amplified at high cell density, conditions under which RAS-3 cells became less sensitive to LY294002-induced VEGF down-regulation. In marked contrast to cells of epithelial origin, ras-transformed murine fibroblasts (3T3RAS) up-regulated VEGF in a manner that was strongly inhibitable by MEK-1 blockade (ie. treatment with PD98059), whereas these cells were relatively unaffected by treatment with the phosphatidylinositol 3'-kinase inhibitor LY294002. In addition, VEGF was up-regulated by 2-3-fold in NIH3T3 cells overexpressing mutant MEK-1. Collectively, the data suggest that the stimulatory effect of mutant ras on VEGF expression is executed in a nonautocrine and cell type-dependent manner and that it can be significantly exacerbated by physiological/ environmental influences such as high cell density.

Asunto(s)

Transformación Celular Neoplásica , Factores de Crecimiento Endotelial/genética , Regulación de la Expresión Génica , Genes ras , Mucosa Intestinal/fisiología , Linfocinas/genética , Neoplasias Experimentales/genética , Neovascularización Patológica , Células 3T3 , Animales , División Celular , Línea Celular , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/patología , Células Epiteliales/fisiología , Fibroblastos/patología , Fibroblastos/fisiología , Flavonoides/farmacología , Mucosa Intestinal/patología , Cinética , Ratones , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Morfolinas/farmacología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Trombospondina 1/genética , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.

Asunto(s)

Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Mesotelioma/genética , Proteoglicanos/genética , Alelos , Animales , Línea Celular , Metilación de ADN , ADN Complementario/análisis , Regulación hacia Abajo , Femenino , Glipicanos , Humanos , Mesotelioma/patología , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344

RESUMEN

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Asunto(s)

Anomalías Múltiples/genética , Trastornos del Crecimiento/genética , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/deficiencia , Factor II del Crecimiento Similar a la Insulina/análisis , Proteoglicanos/deficiencia , Anomalías Múltiples/fisiopatología , Animales , Síndrome de Beckwith-Wiedemann/genética , Síndrome de Beckwith-Wiedemann/fisiopatología , Peso Corporal , División Celular , Femenino , Genotipo , Glipicanos , Trastornos del Crecimiento/fisiopatología , Heparitina Sulfato/genética , Heparitina Sulfato/fisiología , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Túbulos Renales Colectores/anomalías , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/patología , Masculino , Mandíbula/anomalías , Mandíbula/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Fenotipo , Proteoglicanos/genética , Proteoglicanos/fisiología , Síndrome

RESUMEN

Adhesion to the extracellular matrix is a crucial survival signal for epithelial and endothelial cells. Both cell types activate an endogenous death program termed "anoikis" when detached from the solid substratum. The signaling events culminating in anoikis are still unclear; recent studies have implicated Stress Activated Protein Kinase (SAPK), also known as Jun-N-Terminal kinase, as a potentially crucial signal transducer and mediator of anoikis. However, the generality and the causal role of SAPK in anoikis remain unclear and controversial. For these reasons we decided to examine the relationship between induction of anoikis and SAPK activation in three independent cell systems. We report here that in immortalized rat intestinal epithelial cells (IEC-18) and human umbilical vein endothelial cells (HUVEC), SAPK is activated weakly and transiently upon cell detachment while in canine kidney epithelial cells (MDCK) such induction is strong and protracted. However, cell types fail to commit to anoikis after remaining in three-dimensional culture for the time required for complete activation of SAPK. This suggests that there is no temporal correlation between SAPK activation and the onset of anoikis in any of the cell lines studied. We further examined the potential involvement of SAPK in the IEC-18 system by investigating a ras oncogene-transformed variant of IEC-18 cells (IEC-18 Ras 3) which are highly resistant to anoikis. Ras expression did not abrogate activation of SAPK, although these cells do exhibit altered kinetics of SAPK induction upon cell detachment. These results suggest that SAPK is not involved in anoikis regulation and that SAPK activation is likely a cell-type-specific epi-phenomenon.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Muerte Celular , Proteínas Quinasas Activadas por Mitógenos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Línea Celular , Perros , Matriz Extracelular/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Ratas , Transducción de Señal , Factores de Tiempo , Proteínas ras/metabolismo

Asunto(s)

Anomalías Múltiples/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/fisiología , Proteoglicanos/fisiología , Somatomedinas/antagonistas & inhibidores , Anomalías Múltiples/genética , Animales , Glipicanos , Heparitina Sulfato/genética , Factor II del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Ratones , Ratones Noqueados , Proteoglicanos/genética , Síndrome

RESUMEN

Although in vitro anchorage-independent growth is widely used as a marker of cell transformation, the biological implications of this trait are poorly understood. We previously demonstrated that enforced anchorage-independent growth of a nontumorigenic, immortalized epithelial cell line (IEC-18) in multicellular spheroid culture results in massive apoptotic cell death. This death process, termed anoikis, is prevented by expression of transforming oncogenes, which also confer tumorigenic competence. This study examines whether acquisition of an anoikis-resistant phenotype is causally related to the tumorigenic capacity of transformed epithelial cells. Parental IEC-18 cells were subjected to 10 cycles of selection for survival in speroid culture. Unlike parental cells, the resulting anoikis-resistant variants (AR1.10 and AR2.10) formed relatively large tumors in nude mice. Both anoikis-resistant sublines displayed upregulated expression of vascular endothelial growth factor (VEGF), a potent angiogenesis stimulator. VEGF121 overexpression alone did not induce tumorigenic conversion of parental IEC-18 cells, which remained highly susceptible to anoikis. We postulate that both anoikis-resistance and angiogenic-competence contribute to tumor formation. Development of anoikis-resistance can be then viewed as a precondition for expression of the tumorigenic phenotype. Our results suggest that even when angiogenesis is not a rate limiting factor (e.g. in vitro) the selective pressures of solid tumor-like, 3-dimensional growth conditions favoring anoikis resistance result in collateral induction of a proangiogenic phenotype.

Asunto(s)

Apoptosis , Transformación Celular Neoplásica , Mucosa Intestinal/patología , Neovascularización Patológica/etiología , Animales , Línea Celular , Factores de Crecimiento Endotelial/fisiología , Linfocinas/fisiología , Ratones , Ratones Endogámicos BALB C , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.

Asunto(s)

Apoptosis/genética , Apoptosis/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Genes ras , Mucosa Intestinal/metabolismo , Intestinos/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Animales , Cromonas/farmacología , Células Clonales , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Humanos , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Transfección , Proteína Destructora del Antagonista Homólogo bcl-2

RESUMEN

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3-induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line- specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.

Asunto(s)

Apoptosis , Glipicanos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Células 3T3 , Animales , Células COS , Expresión Génica , Glicosilfosfatidilinositoles/metabolismo , Glipicanos/genética , Trastornos del Crecimiento , Células HT29 , Proteoglicanos de Heparán Sulfato/genética , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Proteínas de la Membrana/genética , Mesotelioma , Ratones , Ratas , Síndrome , Células Tumorales Cultivadas

RESUMEN

OCI-5/Glypican 3, a member of the glypican family of proteoglycans, is the defective gene in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 expression is developmentally regulated in the intestinal epithelium, and the mechanism of its regulation was studied in the rat intestinal epithelial cell line IEC-18. A large induction of OCI-5 transcript and protein was observed at high cell density. Among other glypican family members, kappa-glypican also exhibited a confluence-dependent induction in select cell types. Nuclear run-on analysis indicated that cell-density regulation of OCI-5 occurs at the level of transcription. The rat and mouse OCI-5 promoters were cloned and found to be highly conserved, located within CpG islands and contain regions of alternating purine and pyrimidine residues. No TATA-box or recognizable INR element was observed. Consensus binding sites for AP-2, SP-1, zeste and NF-1/CTF are conserved across human, mouse and rat promoters. 5' deletion mapping of the rat promoter identified regions which enhance and repress promoter activity, with no apparent confluence-dependence or tissue-specificity. Nuclear run-on analysis probing different regions of the gene suggests that elongation control plays a role in the induction of OCI-5 by confluence.

Asunto(s)

Regulación de la Expresión Génica , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Proteoglicanos/genética , Animales , Secuencia de Bases , Línea Celular , Glipicanos , Heparitina Sulfato/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ratones , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , Regiones Promotoras Genéticas , Proteoglicanos/metabolismo , Ratas , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Transcripción Genética , Transfección , Regulación hacia Arriba

RESUMEN

CD44 is expressed in various isoforms on numerous cell types and tissues during embryogenesis and in the mature organism. CD44 may also be involved in tumor growth. To study the multiple roles of CD44, we abolished expression of all known isoforms of CD44 in mice by targeting exons encoding the invariant N-terminus region of the molecule. Surprisingly, mice were born in Mendelian ratio without any obvious developmental or neurological deficits. Hematological impairment was evidenced by altered tissue distribution of myeloid progenitors with increased levels of colony-forming unit-granulocyte-macrophage (CFU-GM) in bone marrow and reduced numbers of CFU-GM in spleen. Fetal liver colony-forming unit-spleen and granulocyte colony-stimulating factor mobilization assays, together with reduced CFU-GM in peripheral blood, suggested that progenitor egress from bone marrow was defective. In what was either a compensatory response to CD44 deficiency or an immunoregulatory defect, mice also developed exaggerated granuloma responses to Cryotosporidium parvum infection. Finally, tumor studies showed that SV40-transformed CD44-deficient fibroblasts were highly tumorigenic in nude mice, whereas reintroduction of CD44s expression into these fibroblasts resulted in a dramatic inhibition of tumor growth.

Asunto(s)

Granuloma/patología , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Receptores de Hialuranos/fisiología , Neoplasias Experimentales/fisiopatología , Empalme Alternativo , Animales , Formación de Anticuerpos , Adhesión Celular , Infecciones por Corynebacterium/inmunología , Citotoxicidad Inmunológica , Granulocitos/citología , Ácido Hialurónico/metabolismo , Hipersensibilidad Tardía/inmunología , Inmunidad Celular , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Prueba de Cultivo Mixto de Linfocitos , Macrófagos/citología , Ratones , Ratones Noqueados , Ratones Desnudos , Trasplante de Neoplasias , Linfocitos T Citotóxicos/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virosis/inmunología