Signals transduced by Eph receptors and ephrin ligands converge on MAP kinase and AKT pathways in human cancers.

Eph receptors, the largest known family of receptor tyrosine kinases, and ephrin ligands have been implicated in a variety of human cancers. The novel bidirectional signaling events initiated by binding of Eph receptors to their cognate ephrin ligands modulate many cellular processes such as proliferation, metastasis, angiogenesis, invasion, and apoptosis. The relationships between the abundance of a unique subset of Eph receptors and ephrin ligands with associated cellular processes indicate a key role of these molecules in tumorigenesis. The combinatorial expression of these molecules converges on MAP kinase and/or AKT/mTOR signaling pathways. The intracellular target proteins of the initial signal may, however, vary in some cancers. Furthermore, we have also described the commonality of up- and down-regulation of individual receptors and ligands in various cancers. The current state of research in Eph receptors illustrates MAP kinase and mTOR pathways as plausible targets for therapeutic interventions in various cancers.

Alternative Promoters of GRIK2 (GluR6) Gene in Human Carcinoma Cell Lines Are Regulated by Differential Methylation of CpG Dinucleotides.

The ionotropic glutamate receptor 6 (GluR6 or GRIK2) gene is transcribed by two cell-type-specific promoters in neuronal and non-neuronal cells, which results in five different transcript variants. The purpose of this study was to explore cell-type-specific silencing of these promoters by epigenetic mechanisms. The neuronal and non-neuronal promoter sequences were cloned upstream of the luciferase gene in the pGL3 luciferase reporter vector. Promoter susceptibility to methylation was confirmed by 5-azacytidine and trichostatin treatment, and the status of CpG dinucleotides was determined by bisulfite sequencing of the promoter was determined by bisulfite sequences. GluR6A transcript variant was expressed in the brain, and GluR6B was most abundant in tumor cell lines. The neuronal promoter was methylated in non-neuronal cell lines. The treatment with 5-azacytidine and trichostatin upregulated transcription of the GluR6 gene, and methylation of the GluR6 promoter sequence in the luciferase reporter system led to downregulation of the luciferase gene transcription. Bisulfite sequencing revealed methylation of 3 and 41 CpG sites in non-neuronal and neuronal promoters, respectively. The differential activation/silencing of GluR6 promoters suggests that the transcript variants of GluR6 are involved in tissue-specific biological processes and their aberrant regulation in tumor cells may contribute to distinct properties of tumor cells.

Artesunate-induced Cellular Effects Are Mediated by Specific EPH Receptors and Ephrin Ligands in Breast Carcinoma Cells.

BACKGROUND/AIM: The aberrant regulation of erythropoietin-producing hepatocellular carcinoma (EPH) receptors and ephrin ligands has been implicated in breast carcinoma, and artesunate has been shown to have anticancer effects. The aim of this study was to characterize the involvement of EPH receptors and ephrin ligands in mediating artesunate (ART)-induced growth suppression of normal breast cells and breast carcinoma cell lines. MATERIALS AND METHODS: The normal breast epithelial cells (MCF10A), non-invasive ductal breast carcinoma cells (MCF7), and invasive triple-negative breast carcinoma cells (MDA-MB-231) were grown in the absence or the presence of different concentrations of artesunate. The cells were counted, and total RNA was isolated. The abundance of transcripts corresponding to EPH receptors and ephrin ligands was determined by quantitative polymerase chain reaction. RESULTS: Cell viability was significantly reduced when cells were treated with artesunate, with MDA-MB-231 cells having the highest sensitivity. Artesunate had no significant effect on transcription of EPH/ephrins in MCF10A cells, but markedly increased EPHA8, EPHA10, EPHB6 and ephrin-A2 expression in MCF7 cells, and significantly increased EPHA3 and EPHA10 expression while reducing that of EPHA7 and ephrin-A3 in MDA-MB-231 cells. CONCLUSION: The relative changes in artesunate-treated MCF7 and MDA-MB-231 cells as compared to similarly treated MCF10A cells allow us to implicate combinatorial expression and receptor interactions for EPH receptor-mediated signal transduction that converges into pathways responsible for cell growth, proliferation, and apoptosis. Specifically, the alterations in EPHA7, EPHA8, EPHA10 and EPHB6 transcripts appear to be important participants in artesunate-mediated cellular effects.

Senescence of Normal Human Fibroblasts Relates to the Expression of Ionotropic Glutamate Receptor GluR6/Grik2.

BACKGROUND/AIM: Glutamate receptor GRIK2, previously designated as GluR6, is best described in neuronal cells. However, its biological relevance in non-neuronal cells is not well understood. We have investigated the expression of this important protein in normal human fibroblasts as a function of cell proliferation. MATERIALS AND METHODS: We introduced expression constructs of all five isoforms (A-E) of GRIK2 in normal human fibroblasts and investigated the cells for the presence and localization of GRIK2, as well as for cell proliferation and senescence over a period of 24 days. RESULTS: The expression of GRIK2-A isoform led to immediate cessation of cell proliferation. However, the cell numbers increased by 1.5- to 9.0-fold in 24 days upon transfection with B, C, D and E isoforms, after which they entered a state of senescence. The decreased proliferation was reflected by incorporation of BrdU in only 2-8% of transfected cells even after culturing them for 16 days. CONCLUSION: Our results are indicative of an association between GRIK2 and aging of fibroblasts.

Stem-like Cells from Invasive Breast Carcinoma Cell Line MDA-MB-231 Express a Distinct Set of Eph Receptors and Ephrin Ligands.

BACKGROUND/AIM: Breast cancer cell lines consist of bulk tumor cells and a small proportion of stem-like cells. While the bulk cells are known to express a distinct combination of Eph receptors and ephrin ligands, the transcript profiles of stem-like cells in these cell lines have not been adequately characterized. The aim of this study was to determine Eph receptor/ephrin ligand profiles of cancer stem cells specific to a triple negative breast carcinoma cell line. MATERIALS AND METHODS: The normal breast cell line MCF10A and the invasive breast carcinoma cell line MDA-MB-231 were used to isolate CD24+/CD24- cell populations. The profiles of Eph receptors and ephrin ligands were determined by real-time PCR and the relative abundance in bulk and stem cells were compared. RESULTS: Based on the mean ΔCT values, the descending order of abundance was as follows. Ephrin-A5 > EPHA2 > (EPHA8, EPHB2) > ephrin-B2 > (EPHA7, EPHB4, ephrin-A4) > ephrin-A3 > ephrin-A1 > (EPHB3, ephrin-B1) > EPHA4 > EPHA1 > EPHA10. EPHA6 and ephrin-A2 transcripts were not detectable in stem cells from either cell line. The expression of EPHA4, EPHA7, EPHA8, and ephrin-A5 in MDA-MB-231 stem cells was up-regulated by 12, 20, ~500, and 6.5-fold respectively. CONCLUSION: The up-regulation of transcripts for EPHA8 and its cognate ligand, ephrin-A5, in the stem cells isolated from MDA-MB-231, suggest their involvement in the invasiveness of this cell line. Based on literature reports, we propose the role of EPHA8 and ephrin-A5 in MDA-MB-231 stem cells via the PI3K-AKT-mTOR pathway.

Isoforms of Ionotropic Glutamate Receptor GRIK2 Induce Senescence of Carcinoma Cells.

BACKGROUND: The aberrant regulation of growth and proliferation is a key feature of carcinoma cells. In order to use molecular strategies to correct these defects toward therapeutic purposes, it is important to characterize the entire spectrum of causative molecules. MATERIALS AND METHODS: By using gene transfer technique, SKOV3 ovarian carcinoma cells were transduced with an expression construct of glutamate receptor 6 (glutamate ionotropic receptor kainate type subunit 2, GRIK2) in retroviral vector PQCXIP. The senescence of transduced cells was subsequently characterized. RESULTS: Our results demonstrated that retroviral transduction occurs with high frequency and transduced cells continue to proliferate, albeit at a significantly reduced rate, up to 39 days. Some transduced colonies stopped proliferating after 12 days, and none of the clones proliferated beyond 37 days. The doubling time for these transduced cells increased progressively until they reached a complete cell-cycle arrest. The proliferating cells were distinguished by bromodeoxyuridine incorporation and 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay. The growth and cell cycle arrest in transduced cells accompanied activation of senescence-associated ß-galactosidase. Furthermore, we have demonstrated a decrease in the levels of active protein kinase B and increase in the abundance of inactive cyclin-dependent kinase 1. CONCLUSION: These results indicate involvement of GRIK2 in senescence and suggests GRIK2 as a potential target for therapeutic intervention of cancer cells.

Differential Expression Patterns of Eph Receptors and Ephrin Ligands in Human Cancers.

Eph receptors constitute the largest family of receptor tyrosine kinases, which are activated by ephrin ligands that either are anchored to the membrane or contain a transmembrane domain. These molecules play important roles in the development of multicellular organisms, and the physiological functions of these receptor-ligand pairs have been extensively documented in axon guidance, neuronal development, vascular patterning, and inflammation during tissue injury. The recognition that aberrant regulation and expression of these molecules lead to alterations in proliferative, migratory, and invasive potential of a variety of human cancers has made them potential targets for cancer therapeutics. We present here the involvement of Eph receptors and ephrin ligands in lung carcinoma, breast carcinoma, prostate carcinoma, colorectal carcinoma, glioblastoma, and medulloblastoma. The aberrations in their abundances are described in the context of multiple signaling pathways, and differential expression is suggested as the mechanism underlying tumorigenesis.

Monochromosomal Hybrids and Chromosome Transfer: A Functional Approach for Gene Identification.

Functional complementation of cellular defects has been a valuable approach for localizing causative genes to specific chromosomes. The complementation strategy was followed by positional cloning and characterization of genes for their biological relevance. We herein describe strategies used for the construction of monochromosomal hybrids and their applications for cloning and characterization of genes related to cell growth, cell senescence and DNA repair. We have cloned RNaseT2, GluR6 (glutamate ionotropic receptor kainate type subunit 2-GRIK2) and protein tyrosine phosphatase, receptor type K (PTPRK) genes using these strategies.

EPHA7 and EPHA10 Physically Interact and Differentially Co-localize in Normal Breast and Breast Carcinoma Cell Lines, and the Co-localization Pattern Is Altered in EPHB6-expressing MDA-MB-231 Cells.

Erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors comprise the most abundant receptor tyrosine kinase family characterized to date in mammals including humans. These proteins are involved in axon guidance, tissue organization, vascular development and the intricate process of various diseases including cancer. These diverse functions of EPH receptors are attributed, in part, to their abilities for heterodimerization. While the interacting partners of kinase-deficient EPHB6 receptor have been characterized, the interaction of the kinase-dead EPHA10 with any other receptor has not been identified. By using co-immunoprecipitation, we demonstrated physical interaction between kinase-deficient EPHA10 with kinase-sufficient EPHA7 receptor. Immunocytochemical analyses have revealed that these two receptors co-localize on the cell surface, and soluble portions of the receptors exist as a complex in the cytoplasm as well as the nuclei. While EPHA7 and EPHA10 co-localize similarly on the membrane in MCF10A and MCF7 cells, they were differentially co-localized in MDA-MB-231 cells stably transfected with empty pcDNA vector (MDA-MB-231-PC) or an expression construct of EPHB6 (MDA-MB-231-B6). The full-length isoforms of these receptors were co-localized on the cell surface, and the soluble forms were present as a complex in the cytoplasm as well as the nucleus in MDA-MB-231-PC cells. MDA-MB-231-B6 cells, on the other hand, were distinguished by the absence of any signal in the nuclei. Our results represent the first demonstration of physical interaction between EPHA10 and EPHA7 and their cellular co-localization. Furthermore, these observations also suggest gene-regulatory functions of the complex of the soluble forms of these receptors in breast carcinoma cells of differential invasiveness.

Genetic components in diabetic retinopathy.

Diabetic retinopathy (DR) is a serious complication of diabetes, which is fast reaching epidemic proportions worldwide. While tight glycemic control remains the standard of care for preventing the progression of DR, better insights into DR etiology require understanding its genetic basis, which in turn may assist in the design of novel treatments. During the last decade, genomic medicine is increasingly being applied to common multifactorial diseases such as diabetes and age-related macular degeneration. The contribution of genetics to the initiation and progression of DR has been recognized for some time, but the involvement of specific genes and genetic variants remains elusive. Several investigations are currently underway for identifying DR susceptibility loci through linkage studies, candidate gene approaches, and genome-wide association studies. Advent of next generation sequencing and high throughput genomic technologies, development of novel bioinformatics tools and collaborations among research teams should facilitate such investigations. Here, we review the current state of genetic studies in DR and discuss reported findings in the context of biochemical, cell biological and therapeutic advances. We propose the development of a consortium in India for genetic studies with large cohorts of patients and controls from limited geographical areas to stratify the impact of the environment. Uniform guidelines should be established for clinical phenotyping and data collection. These studies would permit identification of genetic loci for DR susceptibility in the Indian population and should be valuable for better diagnosis and prognosis, and for clinical management of this blinding disease.

Modulation of liver-intestine cadherin (Cadherin 17) expression, ERK phosphorylation and WNT signaling in EPHB6 receptor-expressing MDA-MB-231 cells.

Aberrant expression of erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors has been reported in a variety of human cancer types. In addition to modulating cell proliferation and migration, EPH receptors are also involved in tumor progression. The transcriptional activation and silencing of EPH receptors are also associated with tumorigenesis. However, the mechanisms underlying the involvement of EPH receptors in tumorigenesis have not been completely deciphered. We have investigated and described the role of EPHB6, a kinase-deficient receptor, in modulating the abundance of cadherin 17 and activation of other intracellular signaling proteins. We previously showed that EPHB6 alters the tumor phenotype of breast carcinoma cells. However, the mechanisms underlying these phenotypic changes had not previously been investigated. Herein we demonstrated the downstream effects of EPHB6 expression on the abundance of cadherin 17, mitogen-activated protein kinase (MEK2), extracellular signal-regulated kinase (ERK), phospho-ERK, ß-catenin, phospho- glycogen synthase kinase 3 beta (GSK3ß) (ser21/9), cell morphology and actin cytoskeleton. These comparisons were made between EPHB6-deficient MDA-MB-231 cells transfected with an empty pcDNA3 vector and cells stably transfected with an expression construct of EPHB6. The results indicate elevated levels of MEK2 and phospho-ERK. While there was no change in the amount of ERK, the abundance of cadherin 17, ß-catenin and phospho-GSK3ß was significantly reduced in EPHB6-transfected cells. These studies clearly demonstrate an inverse relationship between the levels of phospho-ERK and the abundance of cadherin 17, ß-catenin and phospho-GSK3ß in EPHB6-expressing MDA-MB-231 cells. From these data we conclude that EPHB6-mediated alterations arise due to changes in abundance and localization of cadherin 17 and activation of WNT signaling pathway. Transcriptional silencing of EPHB6 in native MDA-MB-231 cells and consequent effects on cadherin 17 and WNT pathway may, thus, be responsible for the invasive behavior of these cells.

Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

PURPOSE: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy. METHODS: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups. RESULTS: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin. CONCLUSIONS: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Restoration of senescence in breast and ovarian cancer cells following the transfer of the YAC carrying SEN6A gene located at 6q16.3.

We previously located a senescence gene locus (SEN6A), at chromosome 6q14-21 by a functional strategy using chromosome transfer into immortal ovarian tumor cells. To further elucidate the SEN6A locus, intact chromosome 6 or 6q was transferred into rat ovarian tumor cells and a panel of immortal revertant clones of senescent cells was generated. The panel of independent colonies as well as mixed populations of revertant cells was analyzed for the presence or absence of chromosome 6 specific markers. These investigations led to the identification of a fine deletion of approximately 1cM at chromosomal interval 6q16.3. A contiguous stretch containing five yeast artificial chromosome (YAC) clones was constructed across the deleted region. The non-chimeric YAC clones were retrofitted and transferred into mouse A9 cells by spheroplast fusion to generate YAC/A9 hybrids. YAC DNA present in YAC/A9 hybrids was subsequently transferred by microcell fusion into immortal tumor cells, and the hybrid cells were characterized for their senescence phenotype. Using this functional strategy, the transfer of YAC clone 966b10 was shown to restore senescence in both rat and human ovarian and breast tumor cells. Our results demonstrate that the SEN6A gene is carried on a 1 Mb YAC, 966b10, which maps at 6q16.3.

EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

A paradigm shift in EPH receptor interaction: biological relevance of EPHB6 interaction with EPHA2 and EPHB2 in breast carcinoma cell lines.

EPH receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in axon guidance, tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The varied biological effects of EPH receptors are mediated in part by the expression of these proteins and their intracellular binding proteins. The ability of EPH molecules to form heterodimers within their own class has been suggested, although not exhaustively characterized. We have clarified this phenomenon by showing that EPHB6, a kinase-deficient receptor, can interact with EPHB2 in mammalian cells, and more significantly EPHB6 interacts with EPHA2. However, EPHB6 does not interact with another kinase-deficient receptor, EPHA10. The interaction between EPHB6 and EPHA2 is the first demonstration of an A-type receptor interacting with a B-type receptor. Furthermore, we correlated relative expression of EPHB6, EPHB2 and EPHA2 with non-invasive and invasive phenotypes of breast tumor cell lines. Our results indicate that tumor invasiveness-suppressing activity of EPHB6 is mediated by its ability to sequester other kinase-sufficient and oncogenic EPH receptors. These observations suggest that cellular phenotypes may, in part, be attributed to a combinatorial expression of EPH receptors and heteromeric interactions among the same class, as well as between two classes, of EPH receptors. Our results also suggest that EPHA10 may transduce signals by interacting with other kinase-sufficient receptors in a similar manner.

Tyrosine kinase-deficient EphB6 receptor-dependent alterations in proteomic profiles of invasive breast carcinoma cells as determined by difference gel electrophoresis.

The expression profiles of the erythropoietin producing hepatocellular carcinoma (Eph) receptor family of tyrosine kinases have been previously shown to provide molecular signatures of normal breast cells, breast tumor cells and invasive breast carcinoma cells. In particular, the expression of EphB6 receptor is lost in invasive breast carcinoma cell line MDA-MB-231. The comparative proteomic profiles of native and EphB6-expressing MDA-MB-231 cells using difference gel electrophoresis (DIGE) and liquid chromatography-mass spectrometry of selected proteins are presented in this study. The expression of more than 70 proteins was significantly altered in EphB6-transfected MDA-MB-231 cells. These altered proteins are involved in glycolysis, cell cycle regulation, tumor suppression, cell proliferation, mitochondrial metabolism, mRNA splicing, DNA replication and repair. Although the majority of these proteins have been implicated in tumorigenesis, the impairment of energy homeostasis and altered regulation of signaling pathways appear to be noteworthy targets of EphB6. Based on the identities of altered proteins and the pathways regulated by these proteins, this study suggests that the interactions of EphB6 with a wide variety of proteins lead to altered proteomic profile of EphB6-transfected MDA-MB-231 cells.

Novel spliced variants of ionotropic glutamate receptor GluR6 in normal human fibroblast and brain cells are transcribed by tissue specific promoters.

The members of the ionotropic glutamate receptor family, namely, a-amino-3-hydroxy-S-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors, are important mediators of the rapid synaptic transmission in the central nervous system. We have investigated the splicing pattern and expression of the kainate receptor subunit GluR6 in human fibroblast cell lines and brain tissue. We demonstrate the expression of GluR6A variant specifically in brain, and four variants, namely, GluR6B, GluR6C, GluR6D and GluR6E in fibroblast cell lines. The variants GluR6D and GluR6E have not been described before, and appear to be specific for non-neuronal cells. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter P(N) and non-neuronal promoter P(NN). We have used RNA ligase mediated RACE and in silico analyses to locate two sets of transcription start sites, and confirmed specific transcripts initiated by P(N) and P(NN) in brain cells and fibroblasts, respectively. The domain structure of variants GluR6D and GluR6E revealed the absence of three transmembrane domains. The lack of these domains suggests that the mature receptors arising from these variant subunits may not function as active channels. Based on these structural features in GluR6D and GluR6E, and the observations that GluR6B, GluR6C, GluR6D and GluR6E are exclusively expressed in non-neuronal cells, it is likely that these receptor subunits function as non-channel signaling proteins.

DNA-based assay for EPHB6 expression in breast carcinoma cells as a potential diagnostic test for detecting tumor cells in circulation.

The early detection of breast cancer is critical for improved treatment and better management of the disease. The dissemination of tumor cells into the blood stream is known to occur early in tumor progression and these circulating tumor cells (CTCs) may be detectable before the occurrence of tumor metastasis. Methylation-specific polymerase chain reaction (MSP) can be exploited for detecting CTCs on the basis of differential methylation of numerous gene promoters in normal and carcinoma cells. In this study, we describe the relationship between loss of Ephrin receptor B6 (EPHB6) expression and the aggressiveness of breast carcinoma cell lines (BCCLs). The loss of EPHB6 expression in more aggressive BCCLs is regulated in a methylation-dependent manner. We demonstrate the ability of an EPHB6 MSP to distinguish between methylated and unmethylated EPHB6 promoters, and to predict expression of the EPHB6 transcript and protein. The sensitivity of MSP was related to the volume of blood processed for DNA isolation. As few as 50 tumor cells in 5 ml blood were detectable with a high efficiency. However, the detection of 10 tumor cells/5 ml was not as efficient. On the other hand, 5 tumor cells or 100 pg of free DNA in 200 microl of blood was also easily detectable. Our results suggest that MSP could be applied to detect even a single cell in 1 ml of blood by employing appropriate modifications. The EPHB6 MSP has clinical implications for the prognosis and/or diagnosis of breast and other cancer types including neuroblastoma, melanoma, and non-small cell lung carcinoma wherein EPHB6 expression is lost in more aggressive forms of the disease.

Chromosome 6 encoded RNaseT2 protein is a cell growth regulator.

We have previously shown by chromosome transfer technique that chromosome 6 alters the phenotype of a variety of tumour cells and SV40 immortalized cells. We present here the phenotypic effects of the ectopic expression of RNaseT2, a highly conserved ribonuclease encoded by chromosome 6q27, in SV40 immortalized cell lines. We contrast our findings with those reported for ovarian carcinoma cell lines and an SV40 immortalized cell line transfected with RNaseT2. Although RNaseT2 expression is elevated in normal diploid fibroblasts approaching senescence (passage 64), forced expression of the gene in immortalized cells does not cause them to senesce. A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2. The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2-10-folds in SV40 immortalized cells in response to RNaseT2 expression. Interestingly, some immortalized cells expressed alternatively spliced transcript variants instead of the full-length RNaseT2 transcript. Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

Role of SV40 integration site at chromosomal interval 1q21.1 in immortalized CRL2504 cells.

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by beta-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies.