X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

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.

Activated Ras prevents downregulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal epithelial cells.

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.

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

OCI-5/GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome, induces apoptosis in a cell line-specific manner.

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.

Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

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.

Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells.

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.

Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine.

We report the isolation of a cDNA clone corresponding to a transcript that is accumulated differentially in rat intestine during development. Clone OCI-5 was selected from the rat intestinal cell line IEC-18, which represents primitive intestinal epithelial crypt cells. Expression was high in rat fetal intestine between 15 and 19 days of development and thereafter was progressively down regulated, becoming undetectable after weaning. Clone OCI-5 detected homologous sequences in human and murine cells. In particular, a high level of expression was detected in CaCo-2, a human colon carcinoma cell line, which is known to express molecules characteristic of fetal small intestinal cells. Expression of a homologous gene was also detected in F9 murine teratocarcinoma cells when they were induced to differentiate into parietal or visceral endodermlike cells. When IEC-18 cells were transformed by activated H-ras or v-src genes, expression of clone OCI-5 was suppressed; the degree of down-regulation correlated with the extent of morphological change induced in the transformed IEC-18 cells. The sequence of clone OCI-5 showed an open reading frame that was capable of encoding a protein of 597 amino acids, but no strong homology was found with any of the proteins registered in the protein sequence data base.

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.

Myc potentiates apoptosis by stimulating Bax activity at the mitochondria.

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.

Relationship of c-myc expression to differentiation and proliferation of HL-60 cells.

We have compared changes in c-myc expression in HL-60 promyelocytic leukemia cells induced to differentiate by dimethyl sulfoxide or growth inhibited in an undifferentiated state. Under these conditions, c-myc expression did not correlate with the proportion of proliferating cells. The kinetics of the decrease in c-myc expression upon differentiation induction is paralleled closely by an increasing proportion of histochemically detected differentiated myeloid cells and by a decrease in clonogenic potential but not by changes in the proportion of proliferating cells. Changes in c-myc expression subsequent to differentiation induction can therefore be directly related to the differentiation process rather than to a cell cycle-related phenomenon.

Stability of c-K-ras amplification during progression in a patient with adenocarcinoma of the ovary.

We have identified a case of serous cystadenocarcinoma of the ovary in which the tumor cells display an amplification (from 10- to 20-fold) of the cellular oncogene K-ras. Normal cells purified from the malignant ascites did not show such amplification. Five consecutive samples were obtained by paracentesis over a 9-month period during which the patient received chemotherapy and underwent clinical progression. The level of c-K-ras amplification in the tumor cells did not change during this period. In studies of the tumors of 6 additional patients with adenocarcinoma of the ovary and 5 cell lines of the same histology, we have detected no other example of significant c-K-ras amplification.

Induction of anoikis and suppression of human ovarian tumor growth in vivo by down-regulation of Bcl-X(L).

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.

Isolation of tumor cells from patients with osteosarcoma and analysis of their sensitivity to methotrexate.

Many clinical studies have been conducted on the role of high-dose methotrexate (MTX) in human osteosarcoma, but information about the in vitro effect of MTX on human osteosarcoma cells is lacking. In this paper, the effect of MTX on tumor cells derived from seven patients with osteosarcoma has been studied in an attempt to correlate clinical and in vitro sensitivity to the drug. Isolation of the cells from the primary tumors (four patients) or metastasis (three patients) was carried out with a collagenase treatment followed by purification through a density gradient. The osteosarcoma cells were identified by electron microscopy and histochemical reactions. The cellular sensitivity to MTX was measured by the inhibitory effect of MTX on [3H]deoxyuridine incorporation into DNA. This incorporation ws 50% inhibited in primary tumors at concentrations from 3 X 10(-7) to 3 X 10(-6) M. The metastatic cells isolated from patients that were clinically resistant to high-dose MTX had a 50% inhibition ranging from 1.5 X 10(-7) to 4 X 10(-5) M. Human stimulated lymphocytes, Sarcoma 180 cells, and Ehrlich ascitic mouse cells had a 50% inhibition of about 1.5 X 10(-7) M. When [3H]thymidine incorporation into DNA of human osteosarcoma cells was studied, it was observed that MTX increased its incorporation up to 4-fold. This increase was stable for at lest 6 hr and was only slightly enhanced by the addition of hypoxanthine. The stimulation by MTX of [3H]thymidine incorporation into DNA in stimulated lymphocytes and Ehrlich cells is much smaller, between 40 and 60%. A hypothesis to explain these results is that osteosarcoma cells build their deoxythymidine monophosphate pool largely through the de novo pathway, the salvage pathway being less important. It is suggested that the importance of the de novo pathway for deoxythymidine monophosphate synthesis is a biochemical characteristic of the osteosarcoma cells which could be related to the initial sensitivity of this tumor to MTX and that an activation of the salvage pathway could constitute an additional mechanism of resistance to this drug.

A cell line from a human ovarian carcinoma with amplification of the K-ras gene.

We have recently reported that ascitic cells from a human ovarian carcinoma have a 10- to 20-fold K-ras amplification and that the level of such amplification did not change over a 9-month period during which the patient received chemotherapy and underwent clinical progression. Here we describe an ovarian tumor cell line (HOC-8) which has been derived from that tumor and which also shows a similar level of K-ras amplification. The amounts of K-ras specific mRNA and the Mr 21,000 protein encoded by the amplified gene are correspondingly elevated. karyotypic analysis revealed no detectable double minute chromosomes but did show an abnormal banding region on chromosome 6. This cell line may represent a useful model to investigate the significance of the K-ras gene product for the pathogenesis of human tumors.

Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis.

The growth of solid tumors in vivo beyond 1-2 mm in diameter requires induction and maintenance of an angiogenic response. This can occur through the release of various angiogenic growth factors from tumor cells. One such factor is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a secreted and specific mitogen for vascular endothelial cells. We show that one of the most commonly encountered genetic changes detected in human cancer, i.e., expression of mutant ras oncogenes, is associated with marked up-regulation of VEGF/VPF in transformed epithelial cells. Thus, elevation of the levels of both VEGF/VPF mRNA and secreted functional protein were detected in human and rodent tumor cell lines expressing mutant K-ras or H-ras oncogenes, respectively. Genetic disruption of the mutant K-ras allele in human colon carcinoma cells was associated with a reduction in VEGF/VPF activity. Furthermore, pharmacological disruption of mutant RAS protein function in H-ras transformed rat intestinal epithelial cells by treatment with L-739,749 (a protein farnesyltransferase inhibitor) caused a significant suppression of VEGF/VPF. The results suggest that dominantly acting ras oncogenes may contribute to the growth of solid tumors in vivo not only by a direct effect on tumor cell proliferation but also indirectly, i.e., by facilitating tumor angiogenesis. Hence, pharmacologically targeting mutant ras oncogenes could conceivably suppress solid tumor growth in vivo, in part, by inhibiting tumor-induced angiogenesis.

Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts.

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.

Role of transforming growth factor alpha (TGF-alpha) in the transformation of ras-transfected rat intestinal epithelial cells.

Most intestinal tumors express transforming growth factor alpha (TGF-alpha) while normal immature crypt cells, the targets for tumor initiation, do not. We have used a rat intestinal cell line derived from immature epithelial crypt cells (IEC-18) to investigate the role of this growth factor in intestinal tumorigenesis. ras transformation of IEC-18 cells induces expression and secretion of TGF-alpha. By studying several independently derived IEC-ras clones, we have established that the amount of TGF-alpha secreted is proportional to the level of activated ras expressed by each clone. The growth of all ras-transformed IEC clones is significantly inhibited by neutralizing antibodies against TGF-alpha and by an antisense TGF-alpha expression vector, indicating a mitogenic role for this growth factor through an autocrine loop. To determine whether TGF-alpha itself can transform intestinal cells, IEC-18 clones transfected with TGF-alpha expression vectors were isolated and characterized. None of the TGF-alpha-expressing clones show any signs of tumorigenic transformation even when they secrete as much TGF-alpha as the IEC-ras clones. It seems, therefore, that TGF-alpha per se does not have the capacity to transform rat epithelial intestinal cells and that its role is mostly related to autocrine growth stimulation.

Glypican-3 expression is silenced in human breast cancer.

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.

Overexpression of H-ras oncogene induces resistance to the growth-inhibitory action of transforming growth factor beta-1 (TGF-beta 1) and alters the number and type of TGF-beta 1 receptors in rat intestinal epithelial cell clones.

In this report, we utilize rat intestinal cell (IEC-18) clones expressing an activated human H-ras gene to investigate the relationship between malignant transformation and growth control by transforming growth factor beta (TGF-beta). We demonstrate that clones expressing high levels of H-ras oncogene lose sensitivity to the growth inhibitory action of TGF-beta. The loss of sensitivity is related to the degree of H-ras expression and is shown to be a direct consequence of H-ras expression through the use of a clonal cell line with inducible expression of activated H-ras. Co-incident with the loss of growth inhibition, ras-expressing clones display an altered expression of TGF-beta-binding proteins as detectable by [125I]TGF-beta cross-linking. While IEC-18 cells express type II (92 kDa) binding protein predominantly, H-ras expression induces a shift to predominantly type I (69 kDa) binding protein expression.

Activated ras and src induce CD44 overexpression in rat intestinal epithelial cells.

CD44 is an adhesion molecule that is involved in the progression of several tumor types, including those originating in the intestine. There are several alternatively spliced forms of CD44. Here we show that intestinal epithelial cells express the standard form of CD44 (CD44s). The same form of CD44 is found in IEC-18, a cell line derived from normal rat intestinal crypts. Upon transfection of IEC-18 cells with ras or src, two oncogenes that are frequently activated in intestinal tumors, a significant induction of CD44s is observed. A causal role for ras in this induction is shown by using IEC clones transfected with an inducible ras expression vector. The oncogene-transformed IEC clones display a high degree of hyaluronic acid-dependent cell-cell adhesion that is not observed in the parental IEC-18 cells suggesting that ras- and src-induced overexpression of CD44 can alter the adhesion properties of intestinal cells.