Browsing by Subject "Gene expression."

Now showing 1 - 5 of 5

Open Access
Analysis of differentially expressed geExpression of notch signaling pathway recenes in breast cancer : BRCA1- induced gene expression profiles and meta-analysis gene signature
(2009) Dedeoğlu, Bala Gür
The aim of the first part of this study was to find out the expression profiles of the genes, which were selected from the former BRCA1-induced gene list (OVCA1, OVCA2, ERBIN, RAD21, XRN2, RENT2, SMG1 and MAC30) in normal-matched primary breast tumors and to correlate the gene expression profiles of selected candidate genes with BRCA1 and various pathology parameters. Among the target genes, the expression of ERBIN, SMG1 and RAD21 were found to be highly correlated with that of BRCA1 both in BRCA1 up- and down-regulated cells and this result was validated with qRT-PCR expression profiling of the eight genes in 32 normal-matched primary breast tumor samples. These genes were found to be discriminative between ER(-) and ER(+) tumors as well as grade 1 and grade 3 tumors. Target genes were also analyzed in independent microarray datasets to assess their predictive power for breast tumor grade, subtype and patient survival. ERBIN, SMG1 and RAD21 were found to have predictive roles in these datasets. The aim of the second part of the study was to found appropriate reference genes (RGs) for accurate quantification of target gene expressions in breast tumor tissues. The expression patterns of fifteen widely-used endogenous RGs and three candidate genes that were selected through analysis of two independent microarray datasets were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. 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. Among the eighteen 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. The aim of the third part of this study was to develop a resampling-based metaanalysis strategy. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. A subset of this meta-gene list was shown to predict well-established molecular tumor subtypes, e.g., basal vs luminal or ER+/ER-, with high accuracy and sensitivity based on class prediction analysis of existing breast cancer microarray datasets. Expression of selected genes, tested on 10 independent primary IDC samples and matched nontumor controls by real-time qRT-PCR, supported the meta-analysis results.
Open Access
Analysis of the function of the nuclear matrix-associated protein C1D
(1999) Bilican, Bilada
DNA Double-strand breaks (DSBs) are generated as intermediate stnictures during V(D)J recombination, as a consequence of oxidative metabolism, or can be induced by exogenous factors such as gamma irradiation and radiomimetic drugs. Mutational studies identified the serine/threonine kinase, DNA-PK, as an essential component of DNA DSB repair machinery. The activation of the multi-component DNA-PK complex requires either free DNA ends or an association with the nuclear-matrix associated protein CID, which facilitates the activation of DNA-PK in a DNA end-independent fashion. The activation of DNA-PK through its interaction with CID, joins an increasing body of evidence which suggests a role for higher order nuclear organisation in the orchestration of complex cellular processes such as transcription, RNA splicing, nucleotide excision repair, replication and double-strand DNA break repair. In this study, the yeast two hybrid system was employed to screen a B-cell cDNA library to identify the interacting proteins with CID, and the interactions determined were further characterised. It was found that, CID interacts specifically with the recombinational hotspot binding protein Translin and Translin associated factor X, TRAX, both in vitro and in vivo, providing evidence that C 1D may play a critical role in DNA repair and recombination. Interestingly, an interaction between TRAX and DNAPKcs has also been identified under in vivo conditions. Tlie interaction of TRAX with DNA-PKcs and C ID indicates a connection between DNA double-strand break repair, recombination, and dynamic nuclear architecture.
Open Access
Mechanisms of SSX gene expression regulation at the promoter level
(2009) Dönertaş, Derya
Cancer Testis (CT) Antigen Genes are not transcribed in any of the adult tissues except spermatogonia, oogonia and trophoblasts. This tight regulation of expression is reversed resulting in the reactivation of CT transcription in a wide variety of cancers. CT genes are coordinately expressed and known to be regulated epigenetically. CT genes are reactivated in cancers by a mechanism that leads to the specific hypomethylation of their promoter-proximal sequences. The mechanisms leading to this phenomenon are unknown. The main objective of this thesis was to further unravel the mechanisms regulating CT gene expression at the promoter level. For this purpose, SSX4 ,a typical CT-X gene known to be under the control of a bidirectional promoter, was chosen as a model. We characterized the minimal critical sequences controlling the sense and antisense promoter and discovered a bidirectional promoter with overlapping promoter activities within a 40 bp region. To study how the antisense promoter could mediate sense promoter repression and vice versa, we used two different reporter genes for each of the promoters in a single construct and found that measurable antisense promoter activity was dramatically reduced upon the introduction of a reporter for the sense promoter. The SSX4 antisense promoter is capable of producing a noncoding transcript from the neighboring ornitine aminotransferase-like pseudogene in vivo. This, however, wasn’t confirmed in this study. The possibility of transcriptional interference or the production of a small dsRNA that could affect the regulation of SSX4 gene expression is discussed in the context of the data. Results from experiments where the down-regulation of DICER was studied as a mechanism that could influence CT gene expression are also discussed
Open Access
Monoclonal antibody production for coiled-coil domain containing-124 (CCDC-124) and its molecular characterization
(2010) Gürbüz, İrem
Coiled-coil-domain-containing-124 (CCDC-124) is a novel protein of unknown function. The Ccdc-124 gene is highly conserved among eukaryotic species and is predicted to encode a 223 amino acids long protein. Yeast-two-hybrid assays had shown that CCDC-124 interacts with RasGEF1B guanine exchange factor, which was previously identified as a specific guanine exchange factor for Rap2, a member of the Rap subfamily of Ras-like G-proteins. In order to reveal the cellular function of CCDC-124 protein, different biochemical experiments, including Western Blotting and Sub-cellular localization studies were performed. In those experiments, a polyclonal antibody against an N-terminal peptide of CCDC-124 was used. Nevertheless, due to its polyclonal nature this antibody exhibited non-specific binding. Although it was determined that the gene encodes a 33 kDa protein and that the protein has diffused cytoplasmic localization, the results were not precise. In order to detect the protein more accurately, monoclonal antibodies were generated against CCDC-124. Throughout the project, mice were injected with pure HisTagged CCDC-124 protein. Via hybridoma technology antibodies were generated and selected for their recognition capacities. At the end, 3 positive hybridoma clones were produced: 7F7, 15C11 and 4B3. To characterize the produced monoclonal antibodies, Western Blot experiments were performed and their binding properties were compared to the polyclonal antibody. Among the three monoclones, 4B3 gave the most promising results at 33 kDa, in Western Blotting. The antibodies will be used in the determination of the protein's sub-cellular localization and in the analysis of its response to extracellular signals. These in turn will aid further analyses related to the protein's role within the cell.
Open Access
Novel monoclonal antibodies targeting conformational ERBB2 epitopes
(2012) Ceran, Ceyhan
ERBB2 is a tyrosine kinase receptor which can act as homodimers or heterodimers with other members of the ERBB family. Nearly 30% of breast cancers overexpress ERBB2, which can be effectively targeted by anti-ERBB2 monoclonal antibodies. Trastuzumab directed against an epitope on subdomain IV of the extracellular domain (ECD) of ERBB2 is a clinically used therapeutics but the response rate is poor and acquired resistance is frequent. Pertuzumab that binds to subdomain II and inhibits receptor dimerization is another promising therapeutics under clinical trials. Anti-ERBB2 antibodies directed to novel epitopes are potentially useful tools for replacement and combinatorial therapies. We produced five new anti-ERBB2 antibodies, all directed against epitope(s) present only on the native ECD. They performed selective growth inhibitory effects depending on the level of ERBB2 expression and cellular background. When used alone, novel anti-ERBB2 antibodies displayed modest but significant growth inhibition on SK-BR-3, BT-474 and MDA-MB-361 cells with ERBB2 overexpression; while no detectable inhibition was observed on MCF-7 and T47D cells lacking ERBB2 amplification. When the antibodies were tested in combination with TNF-α, they acted synergistically on SK-BR-3 cells, producing upto 80% growth inhibition; but performed antagonistically on BT-474 cells. Detailed investigation of a representative antibody indicated G1-arrest as the main mechanism of the anti-proliferative effects exerted on SK-BR-3 cells. Antibody treatment induced permanent inhibition of DNA synthesis, leading to accumulation of cells at G1-phase; an effect which was accelerated in the presence of TNF-α. In addition, treated SK-BR-3 cells displayed inhibition of Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest, independently from TNF-α. Novel antibodies against conformational epitopes present on the extracellular domain of ERBB2 receptor may serve as new analytical and diagnostic tools, in addition to being potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-ERBB2 antibodies and TNF-α provide evidence for a complex interplay between ERBB2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of ERBB2-directed therapeutic interventions.