Browsing by Subject "down regulation"

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Open Access
Chitosan polysaccharide suppress toll like receptor dependent immune response
(Turkish Society of Immunology, 2015) Tincer G.; Bayyurt, B.; Arıca, Y.M.; Gürsel İ.
Objectives: Chitosan is a widely used vaccine or anti-cancer delivery vehicle. In this study, we investigated the immunomodulatory effect of chitosan/pIC nanocomplexes on mouse immune cells. Materials and methods: Proliferative and cytotoxic features of chitosan were tested via CCK-8 assay on RAW 264. 7. IL-1β production was assessed via ELISA from PEC supernatants. TNF-α, and NO induction from chitosan treated RAW cells detected by ELISA and Griess assay, respectively. mRNA message levels of TLRs and cytokines on macrophages in response to chitosan/pIC nanocomplex treatments were evaluated by RT-PCR. Results: Results revealed that chitosan is non-toxic to cells, however, proliferative capacities of macrophages were reduced by chitosan administration. Mouse PECs treated with chitosan, led to NLRP3 dependent inflammasome activation as evidenced by dose-dependent IL-1β secretion. Chitosan/pIC nanocomplexes did not improve immunostimulatory action of pIC on RAW cells, since TNF-α and NO productions remained unaltered. Expression levels of several TLRs, CXCL-16 and IFN-α messages from mouse splenocytes were down regulated in response to chitosan/pIC nanocomplex treatment. Conclusion: Our results revealed that chitosan is an anti-proliferative and inflammasome triggering macromolecule on immune cells. Utilization of chitosan as a carrier system is of concern for immunotherapeutic applications. © 2015 Turkish Journal of Immunology.
Open Access
Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues
(Cognizant Communication Corporation, 2009) Gur-Dedeoglu, B.; Konu, O.; Bozkurt, B.; Ergul, G.; Seckin, S.; Yulug, I. G.
Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TTC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRTPCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRTPCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor–normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor–normal paired breast cancer qRT-PCR studies.
Open Access
Imetelstat (a telomerase antagonist) exerts off target effects on the cytoskeleton
(2013) Mender I.; Senturk, S.; Ozgunes, N.; Can Akcali, K.; Kletsas, D.; Gryaznov, S.; Can, A.; Shay J.W.; Dikmen, Z.G.
Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a crucial role in telomere maintenance. This enzyme is expressed in approximately 90% of human tumors, but not in the majority of normal somatic cells. Imetelstat sodium (GRN163L), is a 13-mer oligonucleotide N3'→P5' thio-phosphoramidate lipid conjugate, which represents the latest generation of telomerase inhibitors targeting the template region of the human functional telomerase RNA (hTR) subunit. In preclinical trials, this compound has been found to inhibit telomerase activity in multiple cancer cell lines, as well as in vivo xenograft mouse models. Currently, GRN163L is being investigated in several clinical trials, including a phase II human non small cell lung cancer clinical trial, in a maintenance setting following standard doublet chemotherapy. In addition to the inhibition of telomerase activity in cancer cell lines, GRN163L causes morphological cell rounding changes, independent of hTR expression or telomere length. This leads to the loss of cell adhesion properties; however, the mechanism underlying this effect is not yet fully understood. In the present study, we observed that GRN163L treatment leads to the loss of adhesion in A549 lung cancer cells, due to decreased E-cadherin expression, leading to the disruption of the cytoskeleton through the alteration of actin, tubulin and intermediate filament organization. Consequently, the less adherent cancer cells initially cease to proliferate and are arrested in the G1 phase of the cell cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression. These effects of GRN163L are independent of its telomerase catalytic activity and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion, proliferation and metastatic potential of cancer cells in vivo.
Open Access
PATZ1 is a DNA damage-responsive transcription factor that inhibits p53 function
(American Society for Microbiology, 2015) Keskin, N.; Deniz, E.; Eryilmaz J.; Un, M.; Batur, T.; Ersahin, T.; Atalay, R.C.; Sakaguchi, S.; Ellmeier W.; Erman, B.
Insults to cellular health cause p53 protein accumulation, and loss of p53 function leads to tumorigenesis. Thus, p53 has to be tightly controlled. Here we report that the BTB/POZ domain transcription factor PATZ1 (MAZR), previously known for its transcriptional suppressor functions in T lymphocytes, is a crucial regulator of p53. The novel role of PATZ1 as an inhibitor of the p53 protein marks its gene as a proto-oncogene. PATZ1-deficient cells have reduced proliferative capacity, which we assessed by transcriptome sequencing (RNA-Seq) and real-time cell growth rate analysis. PATZ1 modifies the expression of p53 target genes associated with cell proliferation gene ontology terms. Moreover, PATZ1 regulates several genes involved in cellular adhesion and morphogenesis. Significantly, treatment with the DNA damage-inducing drug doxorubicin results in the loss of the PATZ1 transcription factor as p53 accumulates. We find that PATZ1 binds to p53 and inhibits p53-dependent transcription activation. We examine the mechanism of this functional inhibitory interaction and demonstrate that PATZ1 excludes p53 from DNA binding. This study documents PATZ1 as a novel player in the p53 pathway. © 2015, American Society for Microbiology.
Open Access
A ranking-based meta-analysis reveals let-7 family as a meta-signature for grade classification in breast cancer
(Public Library of Science, 2015) Oztemur, Y.; Bekmez, T.; Aydos, A.; Yulug I.G.; Bozkurt, B.; Dedeoglu, B.G.
Breast cancer is one of the most important causes of cancer-related deaths worldwide in women. In addition to gene expression studies, the progressing work in the miRNA area including miRNA microarray studies, brings new aspects to the research on the cancer development and progression. Microarray technology has been widely used to find new biomarkers in research and many transcriptomic microarray studies are available in public databases. In this study, the breast cancer miRNA and mRNA microarray studies were collected according to the availability of their data and clinical information, and combined by a newly developed ranking-based meta-analysis approach to find out candidate miRNA biomarkers (meta-miRNAs) that classify breast cancers according to their grades and explain the relation between miRNAs and mRNAs. This approach provided meta-miRNAs specific to breast cancer grades, pointing out let-7 family members as grade classifiers. The qRTPCR studies performed with independent breast tumors confirmed the potential biomarker role of let-7 family members (meta-miRNAs). The concordance between the meta-mRNAs and miRNA target genes specific to tumor grade (common genes) supported the idea of mRNAs as miRNA targets. The pathway analysis results showed that most of the let-7 family miRNA targets, and also common genes, were significantly taking part in cancer-related pathways. The qRT-PCR studies, together with bioinformatic analyses, confirmed the results of meta-analysis approach, which is dynamic and allows combining datasets from different platforms. © 2015 Oztemur et al.
Open Access
SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation
(2011) Acun, T.; Oztas, E.; Yagci, T.; Yakicier, M.C.
Background: Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.Methods: SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.Results: We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.Conclusions: Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis. © 2011 Acun et al; licensee BioMed Central Ltd.