Naling pathways in B and T-cells [3,4]. Recent studies revealed that wild-type Vav1, which is normally tightly restricted to hematopoietic cells, is expressed in several human tumor malignancies, suggesting that it has a role in human cancer. The involvement of wild type Vav1 in human tumors was first demonstrated in the neuroblastoma SK-N-MC cell line [5]. A subsequent screen of 42 primary human neuroblastomas revealed that the majority expressed Vav1. Wild-type Vav1 was also identified in more than 50 of 95-pancreatic ductal adenocarcinoma (PDA) specimens MedChemExpress BI 78D3 examined 11967625 and in several PDA cell lines [6]. Patients with Vav1-positive tumors had a worse prognosisthan patients with Vav1-negative tumors [6]. Aberrant expression of Vav1 was also found in over 40 of human primary lung cancers and lung cancer cell lines examined [7] and in melanoma tissue sections and cell lines [8]. Expression of Vav1 was also shown in hematological malignancies such as B cell chronic lymphocytic leukemia (B-CLL), occurring primarily in B-CLL patients with 13q chromosomal deletions [9]. Depletion of Vav1 expression in pancreatic and lung cancer cell lines reduced colony formation in soft agar and tumor size in nude mice. This effect of Vav1 silencing was observed even in the presence of mutant KRas, demonstrating the critical role of Vav1 in tumor development [6,7]. Vav1 might contribute to malignancy by activating signaling cascades through its GEF activity, resulting in cytoskeletal reorganization and transcription [10?2]. Despite its physiological restriction to hematopoietic cells, Vav1 can be phosphorylated on tyrosine residues in cells of other tissue origins following stimulation of growth factor receptors such as EGFR [13], platelet derived growth factor receptor (PDGFR) [14], and the Nerve Growth Factor (NGF) receptor, trk [15]. The additional Vav1-Vav1 in Breast Cancertriggered signaling may overwhelm cellular control mechanisms and promote transformation. To increase our understanding of Vav1 activity and regulation in human cancers, we analyzed the involvement of Vav1 in human breast cancer. In this study, we show that Vav1 is 1655472 expressed in the majority of breast carcinomas and that its ectopic expression in breast cancer cell lines can induce significant changes in these cells, causing either transformation or cell death.Gene ArraymRNA was isolated from cells using the RNeasy mini kit (QIAGEN, Germany), and samples were subjected to GeneChipH Human Exon 1.0 ST Array (Affymetrix, CA, USA). Each sample was composed of a mixture of three independent mRNA isolations. Data was read and RMA normalized using Partek Genomic Suite 6.6. Statistical testing for significant genes and clustering used this package, in addition to dedicated packages written in Matlab R2011A.Materials and Methods Human Breast Tissue ArrayHuman breast paraffin tissue array (http://www.biochain.com/ biochain/datasheet/Z7020004-B410017.pdf) was purchased (Biochain, CA, USA) and treated according to manufacturer’s instructions.RT-PCRTotal RNA and reverse transcription of Vav1 and GAPDH was performed as previously described [7].Quantitative Real-time PCRTotal RNA and cDNAs from cell lines were prepared as above. Detection of Vav1 was performed using cyber green PCR master mix (Tamar, Jerusalem, Israel) and the required Terlipressin supplier primers (Table S1). Analysis was performed using the ABI Prism 7300 real-time PCR technology (Applied Biosystems, CA, USA). Three independent experiments were performed, e.Naling pathways in B and T-cells [3,4]. Recent studies revealed that wild-type Vav1, which is normally tightly restricted to hematopoietic cells, is expressed in several human tumor malignancies, suggesting that it has a role in human cancer. The involvement of wild type Vav1 in human tumors was first demonstrated in the neuroblastoma SK-N-MC cell line [5]. A subsequent screen of 42 primary human neuroblastomas revealed that the majority expressed Vav1. Wild-type Vav1 was also identified in more than 50 of 95-pancreatic ductal adenocarcinoma (PDA) specimens examined 11967625 and in several PDA cell lines [6]. Patients with Vav1-positive tumors had a worse prognosisthan patients with Vav1-negative tumors [6]. Aberrant expression of Vav1 was also found in over 40 of human primary lung cancers and lung cancer cell lines examined [7] and in melanoma tissue sections and cell lines [8]. Expression of Vav1 was also shown in hematological malignancies such as B cell chronic lymphocytic leukemia (B-CLL), occurring primarily in B-CLL patients with 13q chromosomal deletions [9]. Depletion of Vav1 expression in pancreatic and lung cancer cell lines reduced colony formation in soft agar and tumor size in nude mice. This effect of Vav1 silencing was observed even in the presence of mutant KRas, demonstrating the critical role of Vav1 in tumor development [6,7]. Vav1 might contribute to malignancy by activating signaling cascades through its GEF activity, resulting in cytoskeletal reorganization and transcription [10?2]. Despite its physiological restriction to hematopoietic cells, Vav1 can be phosphorylated on tyrosine residues in cells of other tissue origins following stimulation of growth factor receptors such as EGFR [13], platelet derived growth factor receptor (PDGFR) [14], and the Nerve Growth Factor (NGF) receptor, trk [15]. The additional Vav1-Vav1 in Breast Cancertriggered signaling may overwhelm cellular control mechanisms and promote transformation. To increase our understanding of Vav1 activity and regulation in human cancers, we analyzed the involvement of Vav1 in human breast cancer. In this study, we show that Vav1 is 1655472 expressed in the majority of breast carcinomas and that its ectopic expression in breast cancer cell lines can induce significant changes in these cells, causing either transformation or cell death.Gene ArraymRNA was isolated from cells using the RNeasy mini kit (QIAGEN, Germany), and samples were subjected to GeneChipH Human Exon 1.0 ST Array (Affymetrix, CA, USA). Each sample was composed of a mixture of three independent mRNA isolations. Data was read and RMA normalized using Partek Genomic Suite 6.6. Statistical testing for significant genes and clustering used this package, in addition to dedicated packages written in Matlab R2011A.Materials and Methods Human Breast Tissue ArrayHuman breast paraffin tissue array (http://www.biochain.com/ biochain/datasheet/Z7020004-B410017.pdf) was purchased (Biochain, CA, USA) and treated according to manufacturer’s instructions.RT-PCRTotal RNA and reverse transcription of Vav1 and GAPDH was performed as previously described [7].Quantitative Real-time PCRTotal RNA and cDNAs from cell lines were prepared as above. Detection of Vav1 was performed using cyber green PCR master mix (Tamar, Jerusalem, Israel) and the required primers (Table S1). Analysis was performed using the ABI Prism 7300 real-time PCR technology (Applied Biosystems, CA, USA). Three independent experiments were performed, e.
