Identification of genes differentially expressed between benign and osteopontin transformed rat mammary epithelial cells

<p>Abstract</p> <p>Background</p> <p>Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein which has an important role in tumor progression.</p> <p>Findings</p> <p>In this study, we have utilized suppressive subtractive hybridization (SSH) to evaluate OPN regulated gene expression, using the Rama 37 benign non-invasive rat mammary cell line and a subclone, Rama 37-OPN. Rama 37-OPN was produced by stably transfecting Rama 37 with an OPN expression vector and it demonstrates increased malignant properties <it>in vitro</it>. Sequence and expression array analysis of the respective cDNA libraries of over 1600 subtracted cDNA fragments revealed 982 ESTs, 45 novel sequences and 659 known genes. The known up-regulated genes in the Rama 37-OPN library code for proteins with a variety of functions including those involved in metabolism, cell adhesion and migration, signal transduction and in apoptosis. Four of the most differentially expressed genes between the benign and <it>in vitro </it>malignant rat mammary cell lines are tumor protein translationally controlled I (TPTI), aryl hydrocarbon receptor nuclear translocator (ARNT), ataxia telangiectasia mutated (ATM) and RAN GTPase (RAN). The largest difference (ca 10,000 fold) between the less aggressively (MCF-7, ZR-75) and more aggressively malignant (MDA MB 231, MDA MB 435S) human breast cancer cell lines is that due to RAN, the next is that due to osteopontin itself.</p> <p>Conclusion</p> <p>The results suggest that enhanced properties associated with the malignant state <it>in vitro </it>induced by osteopontin may be due to, in part, overexpression of RAN GTPase and these biological results are the subject of a subsequent publication <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Abstract 136: hsa-miR-20a promotes tumorigenesis in ccRCC cancer cell lines

Abstract Objective &amp; Background: We have previously reported specific microRNA profiles in renal cell cancer (RCC) cell lines in vitro (Kurisetty et al. AACR Annual Meeting 2010. A # 3045;). Among the miR signatures identified, miR-20a, a member of 17-92 Oncomir cluster, stood out as a highly upregulated miR in most RCC cell lines, regardless of their VHL status. The objectives of this study are to characterize the biological significance of miR 20A upregulation in RCC. Methods: ccRCC cell lines 786.O, ACHN and KV6 and HREC, were used for cell based biological/phenotypic assays. miRNA-gene target prediction analysis was performed using miRGEN and DIANA lab programs. miR-20a-Mimic (Mi) and miR-20a-Inhibitor (In) were transiently transfected into normal HREC cell line and RCC cell lines, respectively. Cell adhesion, proliferation, softagar colony formation and transwell invasion/migration assays were performed to investigate the influences of miR-20a-Mi/In transfected into normal and ccRCC cell lines. Conditioned medium from transfected cell lines was tested for its ability to induce in vitro angiogenesis in HUVECs. Western blotting was performed to identify the changes in E2F5 protein expression in the miR-20a-Mi/In transfected normal and ccRCC cell lines. Results: In normal HREC cells, upregulation of miR 20a (by miR-20a-Mi), significantly induced cell proliferation, adhesion, migration &amp; invasion and soft agar colony formation. miR-20a knock down in RCC cells (by miR-20a-In) significantly reduced cell proliferation, adhesion, migration &amp; invasion and colony formation to levels similar to non-malignant HREC cells. Prediction analyses through bioinformatics approaches identified E2F5, a transcription factor and a tumor suppressor of the E2F family of transcription factors, as a target of miR-20a. Compared to non-tumor HREC cells, E2F5 was significantly downregulated in RCC cell lines, and miR-20a inhibition enhanced E2F5 expression in cancer cells. miR-20a-Mi enhanced the ability of HREC conditioned medium to induce capillary formation in HUVECs. Conclusion: Our results suggest that miR-20a may promote the tumorigenic properties of RCC in vitro. Moreover, miR 20a may promote angiogenesis. We demonstrated that E2F5 is a target of miR 20a, is reduced in RCC cell lines, and its expression is enhanced by mir-20a inhibition, suggesting that E2F5 may mediate the tumor promoting effects of miR 20a. Molecular mechanisms of miR-20a function are currently being investigated further to understand its tumorigenic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2011-136</jats:p

Fabrication of IR transparent zinc sulphide plate by chemical vapour deposition (CVD)

400-404Recent developments in Chemical Vapour Deposition (CVD) processing with its inherent advantages provide a feasible route for producing high quality optically transparent ceramic materials. CVD process feasibility of the reaction between Zn and H2S in obtaining optical quality ZnS deposits are evaluated with respect to the free energy requirements. Rayleigh number (Ra) of 106 corresponding to laminar flow indicated optimum flow velocities during CVD processing. Deposits show desirable thickness uniformity of 0.55% per cm and were found to have a phase pure ZnS XRD phase. EBSD patterns exhibited a highly oriented columnar ZnS grains and the specimens have shown the theoretical transmission values in the IR ranges

Abstract 4313: Preclinical safety and efficacy of VSV-IFN-b in a syngeneic, immunocompetent model of head and neck squamous cell carcinoma

Abstract Objective&amp; Background: VSV, an RNA virus of the rhabdoviridae family, induces potent cytolytic effects in immortalized cells and cells with defects pathways of IFN or PKR in vitro &amp; in vivo. To take advantage of defects in IFN response in tumors, and to improve the virus’ therapeutic window, “second generation” VSVs expressing IFN-b were developed and characterized (Obuchi et. al. 2003). The aims of this study are to evaluate the effects of VSV-IFN-b in human and rodent head &amp; neck cancer cell lines and to assess the safety and in vivo antitumor efficacy of VSV-IFN-b in syngeneic rat head &amp; neck cancer models. Methods: Rat (FAT-7) and mouse (SCC VII) squamous cell carcinoma cells were infected in vitro with VSV-rat-IFN-b and VSV-mouse -IFN-b. Viruses were propagated using the BHK-21 cells according to established protocols. ELISA &amp; Western blotting were performed to identify the expression of rat-IFN-b post infection, in SCC-VII &amp; FAT-7 cells, respectively. Viral cytotoxicity during normoxia and hypoxia, viral replication and IFN-b expression were assessed to determine sensitivity of rat and mouse SCC cells to VSV-IFN-b. An in vivo model of rat squamous cell carcinoma was established by injection of 3×106/ml of FAT-7 cells SC on the floor of the mouth in immunocompetent female Fisher-344 rats. VSV-rat-IFN-b was administered intratumorally at different doses and schedules when tumors reached a diameter of 70-100 mm3. Antitumor efficacy and toxicity were evaluated during the study. Results: VSV-IFN-b induced significant cytotoxicity and successfully propagated in FAT-7 and SCC VII during normoxia and hypoxia. IFN-b production was increased at 24 and 48 hours after viral infection. Rat SCC cells were more susceptible to VSV-rat-IFN-b than mouse cells, and the cytotoxic effects were similar to VSV-human IFN b on rat cells. Tumors were successfully induced and were palpable at 21 days after inoculation. IT administration of VSV-rat-IFN-b resulted in reduction in tumor size and improved survival compared to the non-treated controls. The antitumor effects of one dose of the virus at 5×108 pfu was more effective than higher doses (5×109) or repeated doses. IV administration of a single dose of VSV-rat-IFN-b (5×108) exerted similar antitumor effects as IT administration. No signs of acute toxicity were observed in treated rats after single or repeated IT or IV virus administration. Conclusion: VSV-IFN-b induces cytotoxicity in FAT-7 and SCC-VII cancer cells during normoxia and hypoxia. In vivo, VSV-rat-IFN-b was associated with antitumor effects and prolongation of survival compared to untreated controls. Characterization of viremia, tissue biodistribution, in vivo viral replication and gene expression in vivo are underway in preparation for future clinical evaluation of VSV-human-IFN-b in subjects with advanced head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2011-4313</jats:p