Control-transfected Cells Research Articles

RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo.

Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5b fl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5b fl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5b fl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

Influence of the DCC gene on proliferation and carcinoembryonic antigen expression in the human colorectal cancer cell line SW1116.

This study investigated the effects of stable transfection of the exogenous wild-type DCC gene on growth of the human colorectal carcinoma cell line SW1116 in vitro. The DCC gene was amplified from normal human colon tissue by reverse transcription-polymerase chain reaction and used to construct a recombinant expression plasmid, pcDNA3.1(+)-DCC. DCC-negative SW1116 cells were transfected with pcDNA3.1(+)-DCC. Cell viability was tested by the methyl thiazolyl tetrazolium (MTT) assay. Immunofluorescence staining was used to determine the effects of pcDNA3.1(+)-DCC on carcinoembryonic antigen (CEA) expression in transfected cells. The number of cells in the population transfected with pcDNA3.1(+)-DCC was lower than in that transfected with the control pcDNA3.1(+) plasmid or in normal cells (t1 = 3.645, P1 < 0.05, t2 = 3.132, P2 < 0.05) at 3-6 days after transfection, and the proliferation rate of pcDNA3.1(+)-DCC transfected cells was also lower (t1 = 2.134, P2 < 0.05; t2 = 2.736, P2 < 0.05). The total viability of pcDNA3.1(+)-DCC transfected cells was lower than that of normal cells (t1 = 3.053, P1 < 0.05) at 2-6 days after transfection, and of control-transfected cells (t2 = 2.816, P2 < 0.05) after 2, 4, 5, and 6 days. The population of pcDNA3.1(+)-DCC transfected colored of green fluorescent cells and their fluorescent intensities were lower than those of control-transfected and normal cells. Therefore, the transfected DCC gene can suppress cell proliferation and lead to downregulation of CEA expression in SW1116 cells, which might weaken its infiltration and metastasis abilities.

Downregulation of microRNA-630 inhibits cell proliferation and invasion and enhances chemosensitivity in human ovarian carcinoma.

MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-23 nt long) that can target genes for either degradation of mRNA or inhibition of translation. miRNAs have not been comprehensively studied in human epithelial ovarian carcinoma (EOC). MicroRNA-630 (miR-630) has been frequently observed to be aberrantly expressed in various types of tumors. The present study explored the functions of miR-630 in the proliferation, apoptosis, chemosensitivity, and invasion of EOC. Using real-time polymerase chain reaction, we detected the miR-630 expression in cancerous, benign, and normal human ovarian tissues. Then, we evaluated the role of miR-630 in cell proliferation, chemosensitivity, apoptosis, and invasion by using the Cell Counting Kit-8, Annexin-V/FITC, and transwell assay on A2780 and SKOV3 cells. Western blotting was performed for analyzing the phosphatase and tensin homolog gene (PTEN) protein expression. The miR-630 expression level was higher in ovarian cancerous tissues than in benign and normal ovarian tissues. Decreased expression of miR-630 suppressed EOC cells' proliferation, migration, and invasion as well as significantly enhanced cell apoptosis and chemosensitivity to cisplatin. Furthermore, PTEN expression was increased in A2780 cells transfected by miR-630 inhibitor in comparison with inhibitor-negative control-transfected cells. In conclusion, downregulation of miR-630 dramatically increased apoptotic cell death chemosensitivity to cisplatin and decreased the proliferation, invasion, and migration of EOC cells. MiR-630 may thus play an important role in the biological behaviors of EOC cells through negative control of the PTEN expression.

CEACAM1 and hollow spheroid formation modulate the chemosensitivity of colorectal cancer to 5-fluorouracil.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) re-expressed and promoted hollow spheroid (HS) formation beyond the invasion front of colorectal cancer. The aim of the present study was to clarify whether CEACAM1 cytoplasmic domain isoform balance and HS are associated with resistance to 5-fluorouracil (5FU). Two-dimensional (D) or 3D culture systems were employed to evaluate the effects of CEACAM1 cytoplasmic isoform balance and HS formation on the chemosensitivity of colorectal cancer cells to 5FU. The risk factors for postoperative recurrence were calculated based on the presence of HS and various clinicopathological characteristics in 82 patients with Stage III colorectal cancer who had undergone curative surgery followed by 5FU-based chemotherapy. CEACAM1-4L-transfected HT29 and CEACAM1-4L and 4S expressing parental LS174T cells had significantly higher resistance to 5FU in comparison with CEACAM1-4S- or vector control-transfected cells. In 3D culture, HS formation induced by CEACAM1-4L induced chemoresistance to 5FU, whereas the solid spheres formed in response to CEACAM1-4S were destroyed by 5FU treatment. HS was identified as an independent factor for recurrence of Stage III colorectal cancer after curative resection followed by 5FU-based chemotherapy. Kaplan-Meier survival curves demonstrated that patients with HS had lower recurrence-free survival rate. CEACAM1 long cytoplasmic domain isoform dominance and HS formation are phenotypes associated with chemoresistance to 5FU.

Functional interaction between cyclooxygenase-2 and p53 in response to an endogenous electrophile

Cyclooxygenase-2 (Cox-2) is rapidly expressed by various stimuli and plays a key role in conversion of free arachidonic acid to prostaglandins. We have previously identified 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived electrophile, as the potent Cox-2 inducer in rat epithelial RL34 cells and revealed that the HNE-induced Cox-2 expression resulted from the stabilization of Cox-2 mRNA that is mediated by the p38 mitogen-activated protein kinase signaling pathway. In the present study, we investigated an alternative regulatory mechanism of Cox-2 expression mediated by a transcription factor p53. In addition, to characterize the causal role for Cox-2, we examined the effects of Cox-2 overexpression in RL34 cells. To examine whether the HNE-induced Cox-2 expression was mechanistically linked to the p53 expression, we analyzed changes in Cox-2 and p53 expression levels in response to HNE and observed that the Cox-2 levels were inversely correlated with the p53 levels. Down-regulation of p53 followed by the activation of a transcription factor Sp1 was suggested to be involved in the HNE-induced Cox-2 gene expression. To characterize the effect of Cox-2 expression in the cells, we established the Cox-2-overexpressing derivatives of RL34 cells by stable transfection with Cox-2 cDNA. An oligonucleotide microarray analysis revealed a dramatic down-regulation of the proteasome subunit RC1 in the Cox-2 overexpressed cells compared to the empty-vector transfected control cells. Consistent with the Cox-2-mediated down-regulation of proteasome, a moderate reduction of the proteasome activities was observed. This proteasome dysfunction mediated by the Cox-2 overproduction was associated with the enhanced accumulation of p53 and ubiquitinated proteins, leading to the enhanced sensitivity toward electrophiles. These results suggest the existence of a causal link between Cox-2 and p53, which may represent a toxic mechanism of electrophilic lipid peroxidation products.

Abstract 2096: HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

Abstract Background The molecular mechanisms of peritoneal dissemination in gastric cancer still remain unclear. This study was designed to clarify the role of HIF-1α in the development of peritoneal dissemination of gastric cancer. Methods HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9 by siRNA transfection. In western blot analysis, HIF-1α expression was assessed using KD and the control transfectant (SC) cells under normoxia (20%O2) as well as hypoxia (1%O2). A series of in vitro analysis were conducted and compared between the 2 transfectants. Using nude mice, peritoneal dissemination was evaluated by orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. Finally, tumor angiogenesis of venous and lymph vessels was immunohistochemically analyzed. Results Deficient HIF-1α expression was observed in KD, but not SC cells under normoxia and hypoxia. In vitro proliferation and invasion was significantly reduced in KD compared with SC cells. On contrary, HIF-1α exerts unfavorable effects on anoikis resistance and adhesion ability. In o.i. model, stomach tumor was highly developed in both of KD (86.7%) and SC mice(100%). On the other hand, peritoneal dissemination was more frequently observed in the SC mice (93%) than in the KD mice (13%) (p &amp;lt; 0.001). In the i.p. model, the dissemination highly occurred in both mice (each 100%), while greater number of nodules was observed in the KD mice (p = 0.017). Angiogenesis with vascular invasion were more highly developed in the SC stomach tumors (venous vessels: p=0.001, lymph vessels: p=0.043). In the disseminated nodules, the vascular invasion was developed at intratumoral regions in the SC o.i. mice, however such appearance was not found in i.p. mice. Conclusions This study clearly showed that HIF-1α is crucial for the development of peritoneal dissemination in the spontaneous metastatic (o.i.) model. Moreover, in i.p. model, larger number of dissemination was found in KD than SC mice, indicating that the dissemination by o.i. would not be developed via seeding mechanism. Analysis of tumor angiogenesis in o.i. model demonstrated that HIF-1α activated venous as well as lymph angiogenesis in the stomach tumor. In peritoneal disseminated nodules, vascular invasion was observed at intratumoral area in o.i. SC mice, but not in the i.p. mice. This unique feature further suggests that the dissemination in o.i. mice might be developed by extravasation of cancer cells, which are micro-connected to the stomach tumor via trans-vessel route. Citation Format: Shuusuke Miyake, Yoshihiko Kitajima, Jun Nakamura, Keita Kai, Kazuyoshi Yanagihara, Tomokazu Tanaka, Masatsugu Hiraki, Kohji Miyazaki, Hirokazu Noshiro. HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2014-2096

Abstract 4369: The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion

Abstract MicroRNA (miR)-218 down-regulation has been reported in numerous human malignancies. In cervical cancer, we identified that lower miR-218 expression was significantly associated with poorer overall survival, disease-free survival, and pelvic/para-aortic lymph node recurrence. Further analyses of cervical cancer data from The Cancer Genome Atlas (TCGA) identified that this down-regulation was associated with a genomic locus loss (hsa-mir-218-1:4p15.31, hsa-mir-218-2:5q34, n=105). The objective of the current study was to elucidate the cellular and molecular functions of miR-218. MiR-218 transfection into cervical cancer cells (SiHa and ME-180) significantly reduced cell migration (by 66% and 89%, respectively), invasion (by 49% and 67%, respectively), and clonogenic capacity (by 42% and 53%, respectively), relative to control-transfected cells (P&amp;lt;0.05). In order to identify clinically relevant miR-218 target genes, we used an integrated trimodal approach, incorporating DNA microarray (Affymetrix Human Genome U133 Plus 2.0) analyses of 79 clinical samples, miR-218 transfection, and miRDB target prediction. The most significant target was survivin (BIRC5); miR-218 transfection confirmed a reduction in survivin mRNA and protein expression in both SiHa and ME-180 cells. Furthermore, a direct interaction between the survivin-3′UTR and miR-218 was validated using a luciferase reporter assay. siRNA knockdown of survivin in SiHa and ME-180 significantly reduced cell migration (by 76% and 83%, respectively), invasion (by 79% and 88%, respectively), and clonogenic capacity (by 98% and 97%, respectively), relative to control cells (P&amp;lt;0.05). YM155, a small-molecule survivin suppressant, effectively reduced survivin mRNA and protein levels in a concentration- and time-dependent manner. This compound correspondingly decreased cervical cancer cell proliferation and clonogenic survival. Our results suggest that the miR-218-survivin axis plays an important role in cervical cancer progression. Citation Format: Ryunosuke Kogo, Christine How, Jeff Bruce, Willa Shi, Kenneth W. Yip, Laurie Ailles, Fei-Fei Liu. The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2014-4369

Gene expression profile analysis of SUDHL6 cells with siRNA-mediated BCL11A downregulation.

Our previous study has shown that downregulation of B-cell chronic lymphocytic leukemia (CLL)/lymphoma11A (BCL11A) gene by small interfering RNA (siRNA) resulted in the growth inhibition and apoptosis of B cell lymphoma cell line SUDHL6. To gain further insight into the molecular mechanisms of this process and identify the differentially expressed genes in SUDHL6 cells after BCL11A downregulation, the global gene expression profile was identified and analyzed using the Affymetrix HG-U133 Plus 2.0 array. Twenty-one differentially expressed genes were validated and analyzed from the BCL11A siRNA-treated SUDHL6 cells. There was a significant dysregulation in the global gene expression of the BCL11A-suppressed SUDHL6 cells. There were 1903 genes differentially expressed with >2-fold changes between the BCL11A siRNA- and negative control-transfected cells. Of these, there were 916 upregulated genes and 987 downregulated genes. The differential genes are involved in various molecular functions and signaling pathways. QRT-PCR validation of the selected differentially expressed genes demonstrated there was a good correlation with the microarray analysis. There was a significant deregulation of expression in the apoptosis-related genes such as BCL-2, BCL2L11 and involved in TGFβ, MAPK, WNT signaling pathways after BCL11A was downregulated in SUDHL6 cells. Our results show that the suppression of BCL11A by RNA interference altered gene expression profile of SUDHL6 cells. The apoptosis-related genes BCL-2, BCL2L11 and the gene alterations in TGFβ, MAPK, WNT signaling pathways might be important in BCL11A siRNA-induced apoptosis of SUDHL6 cells, suggesting BCL11A is involved in gene networks associated with apoptosis.

Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak. In this report, we asked whether the PD-1/PD-L1 pathway has the potential to diminish xenogeneic immune responses also in vivo. L23-PD-L1 or mock transfected control cells (L23-GFP) were transplanted under the kidney capsule of rats. The occurrence of kidney-infiltrating rat leukocytes and the induction of anti-pig antibodies were monitored in grafted animals. Assessment of cellular infiltrates revealed similar numbers of macrophages in kidneys grafted with L23-PD-L1 or L23-GFP control cells. However, the level of MHC class-II molecules was reduced on macrophages responding to L23-PD-L1 grafts, suggesting a lower state of activation. Furthermore, less T cells were found in kidneys receiving L23-PD-L1 cells. In addition, the titers of induced anti-pig antibodies were significantly lower in rats grafted with L23-PD-L1 cells. These data suggest that signals triggered by PD-1-PD-L1 interaction interfere with activation pathways involved in the induction of cellular and antibody-mediated immune responses to xenografts in vivo. Targeting of PD-1 and/or PD-L1 may be a promising approach for immune modulation after xenotransplantation.

MiR-1246 promotes SiHa cervical cancer cell proliferation, invasion, and migration through suppression of its target gene thrombospondin 2.

To investigate the effects of miR-1246 on proliferation, invasion, and migration in the human (CSCC) cell line SiHa. SiHa cells were assigned into three groups: miR-1246 analog; miR-1246 antagonist; and control. The MTT, transwell, and wound healing assays were performed to evaluate the proliferation, invasion, and migration abilities of SiHa cells, respectively. Western blot was carried out to detect protein expression of thrombospondin-2 (THBS2) before and after transfection with miR-1246 analog, antagonist, or control. In addition, a THBS2 3'-UTR-containing dual luciferase plasmid was generated and co-transfected with miR-1246, the inhibitor, or non-specific miRNA, into SiHa cells to observe its effects on THBS2-driven luciferase enzyme activity. MTT, transwell, and wound healing assays revealed that proliferation, migration, and invasion were all significantly enhanced (P<0.01) in SiHa cells transfected with miR-1246 analog, but were suppressed in those transfected with the miR-1246 antagonist. Western blot data showed that miR-1246 analog-transfected SiHa cells had significantly decreased THBS2 expression when compared with control-transfected cells (gray value=6.28±10.22 vs. 9.58±17.58; P=0.013) while those transfected with the miR-1246 antagonist had significantly increased THBS2 expression (gray value=12.90±19.81; P=0.037). Moreover, SiHa cells co-transfected with miR-1246 and the THBS2 3'-UTR-containing plasmid exhibited decreased luciferase enzyme activity compared with the control. MiR-1246 induced CSCC SiHa cell proliferation, invasion and migration. Preliminary evidence suggests that miR-1246 might promote CSCC tumorigenesis and progression by the suppression of its target gene THBS2.

Analysis Of Gene Expression Profile In SUDHL6 Cells Of siRNA-Mediated BCL11A Downregulation

BackgroundThe B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11A gene (BCL11A) was associated with hematopoietic malignancies. However, the precise function of this transcription factor in B-cell malignancies still remain poorly characterized. Previous work from our laboratory has shown that BCL11A gene by small interfering RNA (siRNA) resulted in the growth inhibition and apoptosis of the B cell lymphoma cell line (i.e., SUDHL6 and EB1). The aim of this study was to further elucidate the molecular mechanism of this process by analyzing the gene expression profile in SUDHL6 cells after BCL11A knockdown. MethodsFBCL11A siRNA was transfected into SUDHL6 cells to knock down BCL11A expression. Cells were collected, and RNA isolated for transcriptional profiling using the Affymetrix HG-U133 Plus 2.0 array. The global gene expression profile of the BCL11A siRNA-treated SUDHL6 cells was identified and analyzed. Twenty-one differentially expressed genes were further validated and analyzed from the BCL11A siRNA-treated SUDHL6 cells by real-time quantitative reverse transcript-polymerase chain reaction (qRT-PCR). ResultsFThere were 659 genes differentially expressed between the BCL11A siRNA- and negative control-transfected cells. These included 294 upregulated genes and 365 downregulated genes. The differentially expression genes are involved in various signaling pathways including metabolic pathways, focal adhesion, the MAPK signaling pathway, the cell cycle, the JAK-STAT signaling pathway, the TGF-beta signaling pathway, the WNT signaling pathway, apoptosis, and BCR signaling. qRT-PCR validation of the selected differentially expressed genes demonstrated agreement with the microarray analysis. There was a significant difference in the relative expression level of most of the selected genes differentially expressed between the BCL11A siRNA- and negative control siRNA-treated cells (P<0.05). After the transfection of BCL11A siRNA, among the apoptosis-related genes in the BCL-2 family, BCL2L11 was upregulated 7.24-fold, and BCL-2 was downregulated 3.23-fold. ConclusionsOur results indicate that BCL11A is involved in gene networks with cancer related functions. BCL11A may play a role in gene expression events related to apoptosis. Disclosures:Li:This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

Abstract B022: cDNA microarray analysis of genes associated with reduced annexin A6 expression in invasive breast cancer cells

Abstract Recent studies have shown that the calcium-dependent membrane binding protein, annexin A6 (AnxA6) is involved in cell proliferation, membrane dynamics, calcium fluxes across the plasma membranes and cell-cell and cell extracellular matrix interactions and cell motility. Together with our recent observation that AnxA6 is down regulated in breast cancer, this suggests that AnxA6 may influence the expression of a very diverse set of genes. We therefore, investigated changes in gene expression profiles associated with AnxA6 depletion in invasive breast cancer cells. To do this we generated AnxA6 depleted BT-549 cells with varying extents of AnxA6 down regulation. Two of the cell lines designated BT-A6sh2 and BT-A6sh5 as well as pGIPZ transfected control cells were used in the cDNA microarray analysis. Analysis of the microarrays revealed that a total of 558 genes were significantly down-regulated of which 38 were down regulated by at least 2.0-fold and p &amp;lt; 0.05. Meanwhile, 625 genes were significantly up-regulated and 33 were up regulated by at least 2.0-fold and p &amp;lt; 0.05. Interestingly, the greater the extent of AnxA6 depletion the greater the number of genes that were up or down regulated. Among the up regulated genes were RASGRF2, CDH2, JAG1, PCDH10 and PCDH7. Among the down regulated genes were FNDC1, CD24 SLC4A4, CALB2, CSPG4 and ST8SIA6. Interestingly, LGALS3 and CDH3 were among several other genes that were up regulated while EGFR, a predictive marker of basal-like breast cancer, was among the genes that were down regulated following &amp;gt;80% depletion of AnxA6. Validation of some of these genes by quantitative RT-PCR suggests that AnxA6 influences Ras signaling, cadherin-mediated cell-cell interactions, solute transport, cell-extracellular matrix interactions, calcium signaling and more interestingly, cancer stem cell phenotypes. Down-regulation of CD24 and up-regulation of N- and P-cadherin suggest that reduced AnxA6 expression as previously shown in breast cancer tissues also facilitates the transformation of tumor cells into CD24-low mesenchymal stem cell-like phenotypes. These data suggest that reduced AnxA6 expression in invasive breast cancer is accompanied by modulation of a wide range of genes that influence several aspects of breast cancer progression, some of which are potentially useful targets for prognosis and therapeutic intervention. Citation Format: Amos M. Sakwe. cDNA microarray analysis of genes associated with reduced annexin A6 expression in invasive breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B022.

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation- and proliferation-phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all-trans retinoic acid (ATRA). Quantitative real-time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3-L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty-eight hours after transfection, the messenger RNA expression level of the adipose differentiation-related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)-transfected cells was 3.5% of that in negative control-transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA-transfected cells was 21.8% lower than that in negative control-transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.

Abstract 1479: Modulating AKT induced epithelial mesenchymal transition (EMT) for prevention and treatment of colon cancer.

Abstract Activation of serine threonine kinase-AKT transduces signals that regulate multiple biological processes including cellular proliferation, survival, apoptosis, gene expression, angiogenesis, migration and EMT in many cancer types. Recent studies have shown oncogenic activation of AKT has emerged as a central feature of EMT, endowing the cancer cells with migratory and invasive capability. In our results over expression of AKT in HCT-116 (AKT/HCT-116) (express low levels of pAKT as compared to the other colon cancer cell lines) showed significantly higher cell proliferation and aggressive phenotype with increase in migration and invasion when compared to the vector transfected control cells (pCMV/HCT-116). Further in vivo studies revealed highly aggressive, angiogenic with large tumor volume in AKT/HCT-116 xenografts compared to the control tumors. Down-regulating constitutively active Akt (by a PI3 kinase inhibitor, such as wortmannin or LY 294002) reverses both cell survival and resistance to chemotherapeutic agents, however, these inhibitors causes severe toxicity in animal models. Hence, it is exigent to identify novel molecules, to inhibit Akt signaling, which could be developed as a new therapeutics, as well as preventive agents for colon cancer. Our small molecules screening studies revealed, Withaferin-A (WA) as a potent molecule which significantly inhibited AKT activity that resulted in inhibition of cell proliferation in AKT over expressing HCT-116 cells. At molecular level inhibition of AKT resulted in suppression of its downstream pro-survival signaling such as NFκB and BCL-2 in AKT/HCT-116 cells and pCMV/HCT-6 cells. Oral treatment of WA significantly suppressed AKT-induced tumor growth, invasion and migration in cell culture and xenograft models. These results correlated well at molecular level with significant inhibition of the important EMT markers i.e. snail, slug, β-catenin, MMP-2 and MMP-9. Immunohistochemistry of tumor tissues showed WA treatment resulted in increase in E-cadherin expression with concomitant decrease in the expression and nuclear localization of β-catenin in AKT overexpressing tumors. Decreased levels of pAKT, NFkB, Snail, Slug, vimentin expression were seen in WA treated both pCMV/HCT-116 and AKT/HCT-116 tumors. Interestingly, significant inhibition of micro-vessel formation and length of vessels were evident in WA-treated tumors which correlated with low expression of angiogenic markers like Factor VIII and RETIC. In conclusion, the present study underscores the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in colon cancer cells and WA could be a potent compound for the chemoprevention and/or chemotherapy of colon cancer. Citation Format: Suman Suman, Vittal Kurisetty, Trinath P. Das, Aditi Vadodkar, Ramadevi Subramani Reddy, Rajkumar Lakshmanaswamy, Chendil Damodaran. Modulating AKT induced epithelial mesenchymal transition (EMT) for prevention and treatment of colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2013-1479

Targeting of Kir2.1 and Downregulation of Inward Rectifier K+ Current by miR-212

Downregulation of inwardly rectifying K+ channels contributes to an increased risk of cardiac arrhythmia in heart failure and to impaired cerebral arterial dilation in chronic alcohol consumption. The downregulation mechanism is unknown, although post-transcriptional regulation of gene expression by microRNAs is a strong possibility that has not been fully investigated. miR-212 is markedly augmented in heart failure and in chronic alcoholism, and is predicted by bioinformatic algorithms to target Kir2.1, the predominant inward rectifier K+ channel expressed in heart and arterial smooth muscle. We developed a fluorescence-based assay for identifying microRNA targets, using mCherry red fluorescent protein to report target sequence expression and enhanced green fluorescent protein for microRNA expression. Using this assay, we demonstrated a functional target for miR-212 in the 3′ untranslated region of Kir2.1. Red/green fluorescence intensity ratio was significantly lower in miR-212-expressing HEK293 cells compared to non-targeting control (miR-212 0.72 ± 0.024 (mean ± sem), n = 550; control 1.21 ± 0.025, n = 731; p<.001, log transformed data). The effect of miR-212 was attenuated by mutating the predicted target site (% inhibition 58.0 ± 14.51, n = 3 wild-type; 22.7 ± 1.25, n = 3 mutant). Expression of miR-212 downregulated endogenous Kir2.1 protein in HeLa cells, as shown by quantitative western blot of membrane extracts (band intensity vs Na+/K+-ATPase loading control: miR-212 0.0647 ± 0.0047; non-targeting control 0.0895 ± 0.0045; n=3, p<.05). Endogenous inward rectifier K+ current in HeLa cells was isolated by extracellular application of 100 µM Ba2+ during whole-cell patch-clamp recording. Ba2+-sensitive current density was significantly smaller in miR-212-transfected (n=13) vs control-transfected cells (n=8); p<.01. In conclusion, downregulation of inwardly rectifying K+ current and Kir2.1 expression in heart failure and alcoholic cerebrovascular dysfunction may be functionally linked to upregulation of miR-212.

Competition between NBS1 and ATMIN Controls ATM Signaling Pathway Choice

Ataxia telangiectasia mutated (ATM) protein kinase activation by DNA double-strand breaks (DSBs) requires the Mre11-Rad50-NBS1 (MRN) complex, whereas ATM interactor (ATMIN) protein is required for ATM signaling induced by changes in chromatin structure. We show here that NBS1 and ATMIN proteins compete for ATM binding and that this mechanism controls ATM function. DSB-induced ATM substrate phosphorylation was increased in atmin mutant cells. Conversely, NBS1 deficiency resulted in increased ATMIN-dependent ATM signaling. Thus, the absence of one cofactor increased flux through the alternative pathway. Notably, ATMIN deficiency rescued the cellular lethality of NBS1-deficient cells, and NBS1/ATMIN double deficiency resulted in complete abrogation of ATM signaling and profound radiosensitivity. Hence, ATMIN and NBS1 mediate all ATM signaling by DSBs, and increased ATMIN-dependent ATM signaling explains the different phenotypes of nbs1- and atm-mutant cells. Thus, the antagonism and redundancy of ATMIN and NBS1 constitute a crucial regulatory mechanism for ATM signaling and function.

PpGalNAc T1 as a Potential Novel Marker for Human Bladder Cancer

To investigate the effect of glycopeptide-preferring polypeptide GalNAc transferase 1 (ppGalNAc T1) targeted RNA interference (RNAi) on the growth and migration of human bladder carcinoma EJ cells in vitro and in vivo. DNA microarray assays were performed to determine ppGalNAc Ts(ppGalNAc T1-9) expression in human bladder cancer and normal bladder tissues. We transfected the EJ bladder cancer cell line with well-designed ppGalNAc T1 siRNA. Boyden chamber and Wound healing assays were used to investigate changes of shppGalNAc T1-EJ cell migration. Proliferation of shppGalNAc T1-EJ cells in vitro was assessed using [3H]-thymidine incorporation assay and soft agar colony formation assays. Subcutaneous bladder tumors in BALB/c nude mice were induced by inoculation of shppGalNAc T1-EJ cells and after inoculation diameters of tumors were measured every 5 days to determine gross tumor volumes. ppGalNAc T1 mRNA in bladder cancer tissues was 11.2-fold higher than in normal bladder tissues. When ppGalNAc T1 expression in EJ cells was knocked down through transfection by pSUPER-shppGalNAc T1 vector, markedly reduced incorporation of [3H]-thymidine into DNA of EJ cells was observed at all time points compared with the empty vector transfected control cells. However, ppGalNAc T1 knockdown did not significantly inhibited cell migration (only 12.3%). Silenced ppGalNAc T1 expression significantly inhibited subcutaneous tumor growth compared with the control groups injected with empty vector transfected control cells. At the end of observation course (40 days), the inhibitory rate of cancerous growth for ppGalNAc T1 knockdown was 52.5%. ppGalNAc T1 might be a potential novel marker for human bladder cancer. Although ppGalNAc T1 knockdown caused no remarkable change in cell migration, silenced expression significantly inhibited proliferation and tumor growth of the bladder cancer EJ cell line.

Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation

AbstractAbstract 3872ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro. Disclosures:No relevant conflicts of interest to declare.

Modulation of CXCL‐8 expression in human melanoma cells regulates tumor growth, angiogenesis, invasion, and metastasis

CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.

Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed “primed for death” and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O2 consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O2 consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by “bioenergetics-based profiling” may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.