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1
Wright, Paul Kingsley. „Identification of oestrogen-regulated genes in breast cancer cells“. Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493073.
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Oestrogens are critical agents in the aetiopathogenesis of breast cancer. Oestradiol (E2) acts via the oestrogen receptor (ER) to control the expression of specific genes, the full repertoire of which has not been established.
2
Kao, Ruey-Ho. „Application of differential display technique to breast cancer tissue“. Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342256.
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3
Melkoumian, Zaroui K. „Pharmacological regulation of c-myc gene expression in human breast cancer cells“. Morgantown, W. Va. : [West Virginia University Libraries], 2001. http://etd.wvu.edu/templates/showETD.cfm?recnum=2218.
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Thesis (Ph. D.)--West Virginia University, 2001.Title from document title page. Document formatted into pages; contains x, 152 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 119-149).
4
Chen, Chien-Cheng. „Mechanisms of transcriptional activation of estrogen responsive genes in breast cancer cells“. Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1869.
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5
Lafta, Inam Jasim. „STAG3 gene expression in breast cancer cells“. Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/12329/.
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The expression of cohesin genes has been found to be disregulated in a number of cancers including prostate, breast and squamous cell carcinoma; and mutations in genes that encode cohesin components have been noticed in colorectal cancer and myeloid malignancies (reviewed by Rhodes et al., 2011). It has been suggested that members of the cohesin complex might be considered as a subgroup of cancer biomarkers (Xu et al., 2011a). Therefore, this study focused on studying the expression of STAG genes in breast cancer cell lines and primary breast tumours. More attention was paid for STAG3, because in addition of being the meiotic component of cohesin is also a member of Cancer Testis (CT) antigens. CT antigens have been used successfully as cancer biomarkers as well as therapeutic targets for various malignancies. Our findings show that the STAG1, STAG2 and STAG3 genes are highly expressed at the mRNA level in the breast cancer cell lines including: MCF-7, T-47D, MDA-MB-231 and MDA-MB-468 and primary breast tumours as well compared to normal breast tissue or normal breast cell line, MCF-10A, using qRT-PCR. Interestingly, a tendency for increasing STAG3 mRNA expression was recorded from stage I through stage IV of breast tumour implying that there might be increased expression as the tumour develops. Therefore, STAG3 expression was confirmed at the protein level by immunoblotting, where STAG3 protein bands were produced by all of the studied cancer cells when compared with the normal breast. Jurkat cells were used as a positive control as STAG3 expression in these had been previously established. Further confirmation of STAG3 protein signal was achieved in primary breast tumour tissue sections compared with the normal tissue using immunohistochemistry. Overall, these data suggest that STAG3 may be a suitable novel biomarker for breast cancer detection. Because STAG3 is a potential therapeutic target for breast cancer, RNA interference was successfully used to deplete STAG3 in MCF-7 cells. Analysis of the cell cycle profile by FACS revealed an accumulation of cells in G1 phase, and simultaneous reduction in the number of cells in both S and G2/M phases of the cell cycle. However, when depleting STAG3 using other si-RNAs specific for STAG3, more breast cancer dead cells were reported in MTT toxicity assay compared to MCF-10A. Finally, we studied STAG3 regulation by the transcription factors, E2F4/E2F6, no correlation was found between STAG3 expression and either of E2Fs as depleting any of them did not affect STAG3 expression. Interestingly, we found that RNAi-mediated E2F6 silencing, but not E2F4, in cancer cells caused cell death. On the other hand, MCF-10A cells depleted of E2F6 showed higher survival fraction in MTT. This finding suggests E2F6 as another potential therapeutic target for breast cancer.
6
Eyre, Rachel. „Determining the genes responsible for drug resistance in ovarian and breast cancer stem cells“. Thesis, University of Newcastle Upon Tyne, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.576535.
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The majority of deaths in ovarian and breast cancer are caused by recurrent metastatic disease which is usually multidrug resistant. This progression has been hypothesised to be due in part to the presence of cancer stem cells, a subset of cells which are capable of self renewal and are able to survive chemotherapy and migrate to distant sites. Side population (SP) cells, identified by the efflux of the DNA binding dye Hoechst 33342 through ABC transporters, are a known adult stem cell group and have been suggested as a cancer stem cell in various cancers. The aims of this study were (i) to determine the presence and prevalence of SP cells in ovarian and breast cancer cell lines and clinical samples, and (ii) to ascertain their role both as cancer stem cells and in cancer drug resistance through ABC transporter identification and specific transporter knockdown. SP cells were identified in both ovarian and breast cancer cell lines and clinical samples. These SP cells expressed known stem cell genes and exhibited stem cell characteristics. SP cells in both ovarian and breast cancer cell lines were more drug resistant than non- SP (NSP, bulk tumour cells), and furthermore this drug resistance was shown to be due to expression of different ABC transporters in different tissue specific cancers. ABCG2 was found to be the predominate transporter expressed in breast cancer cell line derived SP populations, however silencing ABCG2 in MCF-7 breast cancer cell SP did not either completely inhibit SP presence or increase cell sensitivity to chemotherapy. In contrast ABCB1 was the predominant transporter expressed in ovarian cancer cell line (IGROV1 and HeyA8MDR) derived SP cells and silencing this transporter both fully inhibited SP cells and significantly increased SP cell death following treatment with paclitaxel. In clinical samples, the presence of SP cells in fine needle aspirates from breast cancer patients correlated to oestrogen receptor negative disease and the triple negative phenotype (ER-,PR-,HER2-), a marker of poor patient prognosis. This study has provided evidence of a role for SP cells in both breast and ovarian cancer. SP cells have a possible prognostic role in breast cancer, and ABCB1 should be considered as a therapeutic target in ovarian cancer.
7
Crook, Simon. „Identification and verification of genes regulated in breast cancer cells by the antiprogestin, Onapristone“. Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250455.
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8
Akhavantabasi, Shiva. „Functional Characterization Of Two Potential Breast Cancer Related Genes“. Phd thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614275/index.pdf.
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Cancer may arise as a result of deregulation of oncogenes and/or tumor suppressors. Although much progress has been made for the identification of such cancer related genes, our understanding of the complex tumorigenesis pathways is still not complete. Therefore, to improve our understanding of how certain basic mechanisms work in normal and in cancer cells, we aimed to characterize two different breast cancer related genes. First part of the study focused on subcellular localization USP32 (Ubiquitin Specific Protease 32) to help understand the function of this uncharacterized gene. USP32 is a member of deubiquitinating enzymes (DUBs) and the gene maps to a gene rich region on 17q23. Genes on 17q23 are known to undergo amplification and overexpression in a subset of breast cancer cells and tumors. DUBs are known to be implicated in a variety of cellular functions including protein degradation, receptor endocytosis and vesicle trafficking. Therefore to elucidate the function of USP32, we localized the full length USP32 protein fused to GFP, in HeLa cells, using Fluorescence Protease Protection (FPP) assay and confocal microscopy. Results suggested a Golgi localization for USP32 as confirmed by co-localization study via BODIPY-TR, a Golgi specific marker. Additional investigations to find the role of USP32 in Golgi will further clarify the function of this candidate oncogene. Second part of the study focused on a potential tumor suppressor. For this purpose, we functionally characterized miR-125b, a microRNA gene as a potential tumor suppressor in breast cancer. microRNAs are regulators of gene expression and their deregulation is detected in cancer cells. miR-125b is reported as a down regulated microRNA in breast cancers. In this study, we investigated the expression, function and possible targets of miR-125b in breast cancer cell lines (BCCLs). Our results revealed a dramatic down regulation of miR-125b in a panel of BCCLs. Restoring the expression of miR-125b in low miR-125b expressing cells decreased the cell proliferation and migration as well as cytoplasmic protrusions, detected by staining of actin filaments. While connection of miR-125b and cell motility based on ERBB2 targeting has been reported earlier, here we present data on ERBB2 independent effects of miR-125b on cell migration in non-ERBB2 overexpressing breast cancer cells. Our results showed involvement of a miR-125b target, ARID3B, in cell motility and migration. Our findings showed miR-125b to be an important regulator of cell proliferation and migration in ERBB2 negative breast cancer cells, possibly through regulating multiple targets.
9
Davis, Jessica. „Identification of novel anti-hormone-induced pro-survival genes in oestrogen receptor-positive breast cancer cells“. Thesis, Cardiff University, 2016. http://orca.cf.ac.uk/98607/.
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The growth inhibitory actions of antihormones in the treatment of oestrogen receptor-positive (ER+) breast cancer are compromised by the development of resistance. There is emerging evidence that antihormones can rapidly induce expression of genes that enable cells to survive the initial impact of these agents and ultimately aid the acquisition of resistance. The aims of this thesis were to identify novel antihormone-induced pro-survival genes in a panel of ER+ breast cancer cell lines and to determine whether such genes contribute to the limited efficacy of antihormones during response and subsequently contribute to the emergence of resistant cell growth. Microarray analysis, together with a stringent filtering process, identified 14 pro-survival genes significantly induced by at least one antihormone treatment (10 day tamoxifen, fulvestrant or oestrogen deprivation) in ER+ MCF-7 breast cancer cells, with increased expression maintained into cell models of antihormone-resistance. Of these 15 genes, 5 (GABBR2, CLU, CTNND2, BCL3 and TSC22D3) were significantly induced by all antihormone treatments. PCR and/or Western blotting demonstrated antihormone-induced expression of these 5 genes in T47D (ER+/HER2-), BT474 and MDA-MB-361 (ER+/HER2+) cell lines. The role of BCL3 and CLU during antihormone response and resistance were next investigated. siRNA-mediated BCL3 knockdown had no effect on cell survival but reduced proliferation of tamoxifen-resistant (TAMR) and oestrogen deprivationresistant (XR) cells. Immunoprecipitation and immunofluorescence studies revealed nuclear localisation and direct association of BCL3 and p50 in TAMR and XR cells. However, during response, BCL3 was located in the nucleus and p50 in the cytoplasm. In contrast, siRNA-mediated CLU knockdown reduced proliferation of fulvestrant-treated MCF-7 cells but was without effect on the growth of resistant cells. To conclude,this thesis has identified one antihormone-induced gene (CLU), which appears to limit response, and a second (BCL3), which appears to promote the growth of antihormone-resistant cells, potentially via activation of NFκB-mediated gene transcription.
10
Wiese, Meike. „Characterisation of HP1γ in mammalian cells“. Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273362.
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The degree of chromatin compaction plays a fundamental role in controlling the accessibility of DNA to the transcription machinery as well as other DNA-dependent biological pathways. The mammalian HP1 (Heterochromatin protein 1) protein family consists of three members: HP1α, β and γ. Each paralogue regulates formation and maintenance of heterochromatin by binding to the repressive chromatin marks H3K9me2/3 with their chromodomains (CDs). Despite high sequence conservation, each HP1 paralogue possesses specific functions, which are likely to be cell type specific. The aim of my thesis was to find novel functions for HP1γ in mouse embryonic stem cells (mESCs) and breast cancer cells. Mass spectrometry analysis identified citrullination of residues R38 and R39 within the CD of HP1γ. I show that these residues are citrullinated by peptidyl arginine deiminase 4 (PADI4) in vitro and in vivo. Mutations in HP1γ (R38/9A), designed to mimic the loss of charge accompanied with citrullination, affect HP1γ’s binding to H3K9me3 peptides and reduce its residence time on chromatin in differentiated mESCs, indicating a role for citrullination in regulating HP1γ binding to chromatin during differentiation. Furthermore, I studied the phenotype of HP1γ depletion in two human breast cancer models and found that HP1γ is essential for cell proliferation and viability of cancer, but not of normal epithelial cells. I performed whole transcriptome analysis in breast cancer cells depleted of HP1γ and cross-referenced it with its genomic localisation, which identified increased expression of interferon/antiviral defense genes and activation of pro-apoptotic pathways. Whilst genes involved in these pathways were not directly bound by HP1γ, this analysis also identified HP1γ as a novel regulator of zinc finger (ZNF) genes. In summary, I identified novel post-translational modifications in HP1γ and characterised them in mESCs. I further demonstrated a role for HP1γ regulating breast cancer cell viability and identified HP1γ as a novel regulator of ZNF genes. My findings highlight HP1γ as a potential target for breast cancer therapy.
11
Uppalapati, Lakshmi Deepthi. „Identification and characterization of unique tumoricidal genes in rat umbilical cord matrix stem cells“. Thesis, Kansas State University, 2010. http://hdl.handle.net/2097/16797.
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Master of ScienceDepartment of Anatomy & Physiology
Masaaki Tamura
Rat umbilical cord matrix stem cells (UCMSC) have been shown to exhibit a remarkable ability to control rat mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in Mat B III growth attenuation, total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in Transwell culture dishes. Gene expression profiles of naive rat UCMSC alone and those cocultured with Mat B III cells were investigated by microarray analysis using an Illumina RatRef- 12 Expression BeadChip. The comparison of gene expression profiles between untreated and cocultured rat UCMSC identified five up-regulated candidate genes (follistatin (FST), sulfatase1 (SULF-1), glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and adipocyte differentiation-related protein (ADRP)) and two down-regulated candidate genes (transforming growth factor, beta-induced, 68kDa (TGFβI) and podoplanin (PDPN)) based upon the following screening criteria: 1) expression of the candidate genes should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) they encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually in co-cultures of 1:20 rat UCMSC:Mat B III cells significantly increased cell proliferation, implying that these gene products are produced under the co-cultured condition and functionally attenuate cell growth. Immunoprecipitation followed by Western blot analysis demonstrated that these proteins are indeed secreted into the culture medium. Individual over-expression of these three genes in rat UCMSC significantly enhanced UCMSC-dependent inhibition of cell proliferation in co-culture. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes, and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumors.
12
Ngwenya, Sharon Khethiwe. „Regulation of E2F-1 gene expression in human breast cancer cells“. Texas A&M University, 2003. http://hdl.handle.net/1969.1/2302.
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17β-Estradiol induces E2F-1 gene expression in ZR-75 and MCF-7
human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells
previously showed that hormone-induced transactivation required interactions
between estrogen receptor α (ERα)/Sp1 bound to upstream GC-rich sites and
NFYA bound to downstream CCAAT sites within the -169 to -54 promoter
region. This promoter region was also E2-responsive in ERα-positive ZR-75
cells; however, further analysis of the promoter showed that cooperative
ERα/Sp1/NFY interactions were not necessary for hormone-induced
transactivation in ZR-75 cells. The upstream GC-rich motifs are activated
independently by ERα/Sp1 in ZR-75 but not MCF-7 cells, and the downstream
CCAAT sites were also E2-responsive. E2 also induced reporter gene activity in
ZR-75 cells transfected with an expression plasmid containing the yeast GAL4
DNA binding domain fused to pM-NFYA and a construct containing five tandem
GAL4 response elements. Subsequent studies showed that hormonal activation
of pE2F-1jm1 and pM-NFYA are dependent on non-genomic pathways in which
E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1
gene expression in ZR-75 and MCF-7 involves different mechanisms,
demonstrating the importance of cell context on transactivation pathways, even
among ER-positive breast cancer cell lines.
TCDD inhibited ERα-mediated responses in MCF-7 and ZR-75 cells. E2-
induced E2F-1protein and mRNA levels in MCF-7 and ZR-75 cells and this
response was inhibited by TCDD. Constructs containing GC-rich sites alone or
in combination with the downstream NFY sites were used in transactivation
studies to investigate the mechanism of inhibitory AhR-ERα crosstalk. Although
TCDD inhibited E2-induced mRNA, protein and reporter gene actitivity, it was
not possible to determine if the inhibitory response was due to limiting ERα
protein levels due to proteasome degradation since proteaome inhibitors alone
blocke hormone-dependent responses. TCDD also inhibited the cAMP/PKA
pathway by inhibiting adenyl cyclase activity. In Drosophila SL-2 cells
cotransfected with the GC-rich -169 to -54 region, ERα and Sp1 plasmids E2
induced transactivation in cells cotransfected with AhR/Arnt expression plasmids
suggesting that the AhR complex suppressed ERα/Sp1 action. These results
demonstrate that TCDD inhibits E2-dependent activation of both non-genomic
and genomic pathways of ER-mediated E2F-1 gene expression.
17β-Estradiol induces E2F-1 gene expression in ZR-75 and MCF-7
human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells
previously showed that hormone-induced transactivation required interactions
between estrogen receptor α (ERα)/Sp1 bound to upstream GC-rich sites and
NFYA bound to downstream CCAAT sites within the -169 to -54 promoter
region. This promoter region was also E2-responsive in ERα-positive ZR-75
cells; however, further analysis of the promoter showed that cooperative
ERα/Sp1/NFY interactions were not necessary for hormone-induced
transactivation in ZR-75 cells. The upstream GC-rich motifs are activated
independently by ERα/Sp1 in ZR-75 but not MCF-7 cells, and the downstream
CCAAT sites were also E2-responsive. E2 also induced reporter gene activity in
ZR-75 cells transfected with an expression plasmid containing the yeast GAL4
DNA binding domain fused to pM-NFYA and a construct containing five tandem
GAL4 response elements. Subsequent studies showed that hormonal activation
of pE2F-1jm1 and pM-NFYA are dependent on non-genomic pathways in which
E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1
gene expression in ZR-75 and MCF-7 involves different mechanisms,
demonstrating the importance of cell context on transactivation pathways, even
among ER-positive breast cancer cell lines.
TCDD inhibited ERα-mediated responses in MCF-7 and ZR-75 cells. E2-
induced E2F-1protein and mRNA levels in MCF-7 and ZR-75 cells and this
response was inhibited by TCDD. Constructs containing GC-rich sites alone or
in combination with the downstream NFY sites were used in transactivation
studies to investigate the mechanism of inhibitory AhR-ERα crosstalk. Although
TCDD inhibited E2-induced mRNA, protein and reporter gene actitivity, it was
not possible to determine if the inhibitory response was due to limiting ERα
protein levels due to proteasome degradation since proteaome inhibitors alone
blocke hormone-dependent responses. TCDD also inhibited the cAMP/PKA
pathway by inhibiting adenyl cyclase activity. In Drosophila SL-2 cells
cotransfected with the GC-rich -169 to -54 region, ERα and Sp1 plasmids E2
induced transactivation in cells cotransfected with AhR/Arnt expression plasmids
suggesting that the AhR complex suppressed ERα/Sp1 action. These results
demonstrate that TCDD inhibits E2-dependent activation of both non-genomic
and genomic pathways of ER-mediated E2F-1 gene expression.
13
Bankole, Habeeb Adebodun. „In silico and molecular validation of identified putative genes and functional analysis of a N K G2D ligand as a breast cancer biomarkers“. University of the Western Cape, 2015. http://hdl.handle.net/11394/4864.
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Philosophiae Doctor - PhDThe current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data.
14
Lu, Shan. „Characterization, Epigenetic Drug Effect, and Gene Delivery to Breast Cancer Cells“. University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1447718189.
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15
Motevalli, Azadeh. „Expression of shelterin and shelterin-associated genes in breast cancer cell lines“. Thesis, Brunel University, 2014. http://bura.brunel.ac.uk/handle/2438/8492.
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Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. The objective of this project was to investigate whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. Initial findings showed that most of the Shelterin and Shelterin-associated genes were significantly down-regulated (at the mRNA expression level) in a panel of ten breast cancer cell lines. Epigenetic alterations to DNA (methylation at CpG Islands) and histones can result in altered expression of genes. Further investigations showed that the promoter region of POT1 was partially methylated in the breast cancer cell line, 21NT. To support these observations, a DNA methylation inhibitor, 5-aza-CdR, and a histone deacetylation inhibitor, TSA, were used in an attempt to reactivate the expression of silenced genes. This work generated novel findings. Treatment with 5-aza-CdR and TSA resulted in the highest recovery of TIN2 and POT1 mRNA levels at both short-term (48 and 72 hours) and long-term (3 weeks) treatment of the breast cancer cell line, 21NT cells. In addition, POT1 promoter methylation was analysed before and after treatment of 21NT cells. Bisulphite sequencing data were consistent with the mRNA expression results, showing up-regulation of POT1, as all methylation sites were demethylated after the treatment of 21NT cells with 5-aza-CdR. These studies also showed for the first time that both the short-term (72 hours) and 3 weeks treatment of 21NT cells with 5-aza-CdR was able to increase telomere lengths (using four measurement methods, i.e. TRF, q-PCR, flow-FISH and iQFISH). Breast cancer cell lines expressed low levels of several telosomal mRNAs and that this down-regulation was found to be due in part to promoter methylation. Methylation was shown to be relieved through treatment of the cells with 5-aza-CdR and TSA; specifically, POT1 was shown to be up-regulated to a higher extent compared with other Shelterin genes. Given that previous studies involved over-expression of POT1 in telomerase-positive cells to demonstrate telomere length elongation, we addressed the possibility that over-expression of POT1 may affect telomere length in 21NT breast cancer cells. The results showed that the average telomere length of the POT1 over-expressing clones was increased by 2 to 3 kb compared with 21NT non-transfected and empty vector controls. The study also demonstrated that increased telomere length (by ectopic over-expression of POT1) is not due to a direct effect of telomerase enzyme activity. One explanation for this could be that POT1 may induce a negative regulator of telomerase activity to maintain telomere length. Taken together, the results generated in this project suggest that POT1 may control a localised activation of telomerase enzyme at the telomere end, and regulate stability of the Shelterin complex.
16
Calvo, Vidal Verónica Alejandra. „Searching for a functional relationship between the breast cancer susceptibility gene BRCA1 and the progesterone receptor in breast cancer cells“. Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7239.
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Germ-line mutations in the breast cancer susceptibility gene BRCA1 strongly increase the risk of developing breast and ovarian cancer in women. Different hypothesis have been proposed to explain this tissue specificity. One of the most argued hypothesis is the one that proposes a link between BRCA1 and ovarian hormones' action. Much data have been published in the last years pointing to an important role of progesterone receptor (PR) in inducing normal mammary development and also breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have found that BRCA1 inhibits the transcriptional activity of PR. We have investigated in more detail the mechanism of this effect. BRCA1 and PR interact in vivo in a ligand-independent fashion. Most importantly, BRCA1 alters the ligand-independent and dependent degradation of PR protein through its ubiquitination and this might have a direct effect on the level of PR recruitment on regulated promoters. BRCA1 is recruited to the hormone-responsive regions of PR-target genes and affects the presence of histone deacetylase activity and the level of monoubiquitinated histone H2A, linking BRCA1 action with chromatin status. These findings support a connection between BRCA1, the principal tumour suppressor responsible for familial breast cancer, and the progesterone receptor transcriptional activity. This relationship can be hypothesized to be reflected in the BRCA1-related breast tumourigenesis.Mutaciones germinales en el gen breast cancer susceptibility gene BRCA1 aumentan altamente el riesgo de padecer cáncer de mama y ovario en mujeres. Se han propuesto diferentes hipótesis para explicar esta especificidad de tejido. Una de las hipótesis más argumentadas es la que propone una relación entre BRCA1 y la acción de las hormonas ováricas. En los últimos años se han publicado numerosos datos señalando al papel esencial del receptor de progesterona (PR) en la inducción del desarrollo normal de la mama y en la formación del cáncer de mama. Este estudio pretendía buscar una relación funcional entre BRCA1 y PR en células de cáncer de mama. Hemos demostrado que BRCA1 inhibe la actividad transcripcional de PR. Hemos investigado en más detalle el mecanismo de este efecto. BRCA1 y PR interaccionan in vivo de una manera independiente de ligando. Y lo que es más, BRCA1 altera la degradación independiente y dependiente de ligando de PR a través de su ubiquitinización y esto podría tener un efecto directo en el nivel de reclutamiento de PR en promotores regulados. BRCA1 es reclutado a las regiones de respuesta a hormona de genes diana de PR y afecta la presencia de actividad histona desacetilasa y el nivel de histona H2A monoubiquitinada, estableciendo un enlace entre la acción de BRCA1 y el estado de la cromatina. Estos hallazgos apoyan una conexión entre BRCA1, el principal supresor de tumor responsable del cáncer de mama hereditario, y la actividad transcripcional del receptor de progesterona. Se puede hipotetizar que esta relación se ve reflejada en el proceso de tumorigénesis BRCA1-dependiente.
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Egwuekwe, Ejike Roland. „Vitamin D Receptor Gene Polymorphisms Knowledge And Breast Cancer In Texas“. ScholarWorks, 2019. https://scholarworks.waldenu.edu/dissertations/6199.
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Breast cancer is a world health problem and is a leading cause of cancer-related death among women in the United States. However, breast cancer risks were reported to be reduced through exposure to Vitamin D through its Receptors identified as the p53 target gene. The purpose of this study was to assess the associations between VDR gene polymorphisms knowledge/awareness and decisions to reduce breast cancer risks and likelihood of mammogram screening among women in Texas. Data from survey were used. Roy adaptation model was the theoretical framework that guided this quasi- experimental, quantitative research. The dependent variables were decisions to reduce breast cancer risks and likelihood of mammogram screening. The independent variables were knowledge about VDR gene polymorphisms and exposure to vitamin D. The covariates were level of education, awareness, lifestyle, breast self-exams, mammograms, age, early menarche, late menopause, and family history of breast cancer. The chi-square test and regression analysis were used to test the stated research hypotheses and to answer the research questions. Knowledge of VDR gene polymorphisms and exposure to vitamin D were not significantly associated with breast cancer risk, ï?£2 (3, N= 250) =3.84, p > 0.05. Also, awareness of the risk factors for breast cancer was not significantly associated with decisions to go for mammogram screenings or to enroll in breast cancer risk-reduction programs, ï?£2 (3, N= 250) =1.58, p > 0.05. To advocate for the promotion of awareness of the importance of pharmacogenetic testing for VDR gene polymorphisms for early detection of breast cancer, which would help to undertake appropriate therapeutic measures in a timely manner to prevent cancer metastasis, further research is warranted.
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Smith, David John. „Transcriptional regulation of the human cathepsin D gene in breast cancer cells“. Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320396.
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Slahudeen, Sameera. „Red palm oil as a therapeutic agent in triple-negative breast cancer patients“. University of the Western Cape, 2020. http://hdl.handle.net/11394/8094.
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Magister Scientiae (Medical Bioscience) - MSc(MBS)Purpose: Breast cancer is one of the most frequent and fatal diseases women all around the globe are challenged with today. In women, breast cancer has the highest mortality rate of all cancers and the incidence rate is on the increase. It is estimated that by the year 2025, 19.3 million women will become a victim of this grave health problem. This disease is a result of the formation of malignant tumours caused by genetic alterations that are involved in the proliferation of cells, cellular differentiation and the disturbance in homeostasis which subsequently leads to the abnormal multiplication and growth of cells. Breast cancer is considered a multifactorial disease with various risk factors such as age, radiation exposure, hormone therapy, oral contraceptives, dietary factors, environmental exposure and genetic predispositions. Breast cancers can be subdivided and classified based on their cellular surface receptors such as Estrogen Receptors, Progesterone Receptors and Human Epidermal Growth Factor Receptor 2. Of the various subtypes, the triple-negative breast cancer subtype which is negative for all 3 surface receptors and presents as the most aggressive form of breast cancer with a poor prognosis. Between 10-20% of all breast cancer cases are classified as triple-negative breast cancer. Due to the hormonal status of triple-negative breast cancer, treatment options are limited and thus of great concern. Chemotherapy remains the most common treatment modality, but prognosis is poor with relapse within years ultimately leading to poor survival outcome. Due to this lack of effective treatment plans, an alternative treatment with minimal side effects and better survival remains an imperative area to explore. A wide scope of literature highlights red palm oil and its health benefits, with its growth inhibitory potential drawing great attention. Red palm oil, extracted from the Elaeis guineensis palm tree is red in colour due to the abundance of carotenoids, tocotrienols and tocopherols found in the oil. Various compounds make up the oil such as lycopene, carotenes, vitamin E and coenzyme Q10. Most studies have researched the effects of vitamin E extracted from the oil as a contributor to its growth inhibitory activity. This study focuses on the effects of the commercial red palm oil as a whole with all its compounds on the proliferation of breast cancer cells as well as the effect it has on various genes associated with breast cancer. Method: This study investigated the effect of red palm oil concentrations (1, 10, 100, 500 and 1000 μg/ml) on breast cancer cells—MCF-7 and MDA-MB-231 with comparison to a non-cancerous cell line—MCF-12A for 24-, 48- and 72-hour treatment periods. The parameter investigated was cell proliferation through the CCK-8 cell proliferation assay and the morphology following red palm oil treatment was observed and captured. Additionally, this study also investigated the effect of red palm oil on the expression of Human Mammaglobin (hMAM) and Maspin genes through the PCR assay and results visualised through agarose gel electrophoresis. Data was statistically analysed using GraphPad version 6.0 software. Results: Following treatment of red palm oil, no apparent changes in the cell morphology was observed despite using variable treatment concentrations over variable times for MCF-7, MDA-MB-231 and MCF-12A cells relative to their respective controls. Immortalised MCF-12A cells showed a significant increase in proliferation with the varying treatment concentrations, but more prominently with the highest concentration at 24, 48 and 72 hours. MCF-7 cells showed significant decreases at 24 and 72 hours. Decreased proliferation was observed at all dosages used, particularly at 10, 100, and 500 μg/ml. Furthermore, MDA-MB-231 cells demonstrated a gradual increase in cell proliferation for the 3 selected time periods in the varying concentrations. Additionally, red palm oil did not alter the gene expression of Maspin at any of the varying treatments for MDA-MB-231 nor MCF-7 cells. However, changes in hMAM gene expression were observed at treatment concentration of 100 μg/ml in MDA-MB-231 cells that were incubated for 24 and 48 hours. However, the hMAM expression was not affected in treated MCF-7 cells. Conclusion: Red palm oil, as an alternative dietary oil, seems to have potential growth inhibitory properties as demonstrated by the change in the cell proliferation of the MCF-7 cells. Literature show that various individual compounds extracted from red palm oil have anti-proliferative and inhibitory effects on breast cancer cells making them good candidates for therapy. However, this study concludes that red palm oil as a whole component would not be a suitable therapeutic agent for highly aggressive triple-negative breast cancer.
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Wierer, Michael 1982. „Role of PLK1 in steroid hormone signaling in breast cancer cells“. Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/283474.
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Steroid receptors (SRs) are hormone-induced transcription factors that regulate gene expression by their concerted actions with coregulatory proteins. Performing a quantitative mass spectrometry-based interaction screen in breast cancer cells, we identified Polo-like kinase 1 to interact with progesterone receptor (PR) and estrogen receptor (ER) in presence of their respective hormone stimuli. PLK1 is recruited to chromatin and regulates SR-dependent and –independent gene transcription. Global gene expression analysis let us observe that PLK1-coactivated genes are enriched in developmental functions and linked to good clinical prognosis in breast cancer patients. Using large-scale phosphoproteomics with MCF7 breast cancer cells treated with estradiol in presence or absence of the specific PLK1 inhibitor BI2536, we observed that 28% of estradiol-induced phosphorylation sites depended on PLK1 activity. Among proteins with PLK1-dependent phosphorylation sites, we identified several transcriptional regulators including the ER-associated histone H3K4-methylase MLL2. As PLK1 inhibition reduced the estradiol-mediated induction of H3K4 methylation levels at MLL2 target genes, together these results propose a role of PLK1 in the regulation of MLL2 activity. Finally, in a targeted approach using recombinant histone proteins as substrate of PLK1, we observed a specific phosphorylation of histone H2B at serine 36. In summary, we conclude that PLK1 affects SR-dependent and SR-independent gene transcription by phosphorylating several transcriptional regulators and chromatin components.
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Clayton, Simon James. „Regulation of oestrogen receptor and oestrogen responsive genes by insulin, IGF-I, oestrogen and antioestrogens in breast cancer cells“. Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283743.
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Ohta, Naomi. „Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes“. Thesis, Kansas State University, 2013. http://hdl.handle.net/2097/16730.
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Master of ScienceDepartment of Anatomy and Physiology
Masaaki Tamura
Previous studies have shown that both human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analysis of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Accordingly, the present study was designed to clarify their different tumoricidal abilities by analyzing gene expression profiles in two types of UCMSC. Gene expression profiles were studied by microarray analysis using Illumina HumanRef-8-V2 and RatRef-12 BeadChip for the respective UCMSC. The gene expression profiles were compared to untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells; human UCMSC vs. MDA-231 human carcinoma cells and rat UCMSC vs. Mat B III rat carcinoma cells. The following selection criteria were used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability; 1) gene expression difference should be at least 1.5 fold between naive UCMSC and those co-cultured with breast carcinoma cells; 2) they must encode secretory proteins and 3) cell growth regulation-related proteins. These analyses screened 17 common genes from human and rat UCMSC. The comparison between the two sets of gene expression profiles identified that two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. The suppression of either protein by the addition of a specific neutralizing antibody in co-culture of rat UCMSC with Mat B III cells significantly abrogated UCMSC ability to attenuate the growth of carcinoma cells. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their 4 ability to suppress the growth of MDA-231 cells. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that both ADRP and FST may play important roles in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.
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Khan, Shaheen Munawar Ali. „Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells“. Texas A&M University, 2005. http://hdl.handle.net/1969.1/4935.
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The CAD gene is trifunctional and expresses carbamoylphosphate
synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for
pyrimidine biosynthesis. CAD gene activities are induced in MCF-7 human breast cancer
cells, and treatment of MCF-7 or ZR-75 cells with 17b-estradiol (E2) resulted in a 3-5
fold increase in CAD mRNA levels in both cell lines. E2 induced reporter gene activity
in MCF-7 and ZR-75 cells transfected with a construct containing the growth-responsive
-90/+115 (pCAD1) region of the CAD gene promoter, which contains three upstream
GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD
gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required
for E2-responsiveness. Results of gel shift and chromatin immunoprecipitation (CHIP)
assays show that both Sp1 and estrogen receptor a (ERa) interact with the GC-rich
region of the CAD gene promoter. Moreover, hormone-induced transactivation of
pCAD1 was inhibited by cotransfection with dominant-negative Sp1 expression plasmid
and small inhibitory RNA for Sp1. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the CAD gene is also
regulated by a nonclassical ERa/Sp1-mediated pathway.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon
receptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and
mammary tumors and in human breast cancer cells. TCDD inhibited hormone-induced
activation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 cells
transfected with E2-responsive pCAD promoter constructs. E2-mediated transactivation
of pCAD constructs with a mutant inhibitory dioxin responsive element DRE (iDRE)
were also inhibited by TCDD suggesting that inhibitory AhR-ERa/Sp1 crosstalk was
iDRE-independent. It was not possible to determine whether the levels of ERa in cells
cotreated with E2 plus TCDD were limiting since the proteasome inhibitor MG132 itself
directly decreased CAD mRNA levels. Using fluorescence resonance energy transfer
(FRET), it was shown that both E2 and TCDD enhanced AhR-ERa interactions. E2 also
induced interactions between ERa and Sp1. However cotreatment with TCDD abrogated
this effect. Results of this study demonstrate a unique model of AhR-ERa crosstalk
where the liganded AhR inhibits ERa-Sp1 interactions and also recruits ERa to Ahresponsive
gene promoters such as CYP1A1.
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Christensen, Kimberly Laura. „The developmental regulator SIX1 plays multiple roles in breast cancer initiation and progression /“. Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 115-132). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Park, Jung. „The transcription factor activator protein family of genes in mammary gland development and breast cancer progression“. Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5596.
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Breast cancer is currently the second most common form of cancer and the second leading cause of death due to cancer in the United States. Breast cancer itself is subdivided into at least four subtypes, luminal A, luminal B, HER2-enriched, and basal-like, based on genomewide molecular expression patterns. Luminal A is the most common form and typically characterized by high levels of estrogen receptor (ER). HER2-enriched cancers usually, but not always, harbor amplified copies of the HER2 oncogene. Luminal B cancers share characteristics with the luminal A and HER2-enriched subtypes. Finally, basal-like cancers are more oftentimes defined by their lack of any markers or molecular targets. Thus, they are often called triple-negative breast cancer. Recent evidence suggests that there are a number transcription factors that play critical roles in the cancer progression of these malignancies. Indeed, TFAP2C has been clearly shown to positively regulate ER in luminal A cancers. Alternatively, TFAP2A appears to play an interesting, but as of yet incompletely, understood role in basal-like cancer. There has been additional evidence that suggests TFAP2C regulates multiple members of the ErbB family of receptor tyrosine kinases. Thus, we hypothesize that the TFAP2 family of transcription factors play a critical role in breast cancer progression. More specifically, we will show that TFAP2A and TFAP2C not only regulate a few critical genes in luminal and basal-like cancer, but instead are responsible for the genomewide expression pattern of these two breast cancer subtypes. Moreover, we argue that TFAP2C's regulation of certain receptor tyrosine kinases in luminal A cancers indicates promising therapeutic targets, particularly with small molecule inhibitors that are already FDA-approved. In addition, we provide data suggesting that TFAP2C likely plays an oncogenic role in HER2-positive breast cancer, possibly through the regulation of certain members of the ErbB family of receptor tyrosine kinases, such as EGFR. To address these points, we use a combination of genetically engineered mouse models, xenografts, siRNA mediated knockdown technology, western blot, qPCR, and number of additional molecular biological techniques. These results will not only establish the family of TFAP2 family of proteins as critical regulators of cancer progression, but our findings will specify how and to what extent each subtype of breast cancer is affected by individual members of the TFAP2 family of transcription factors.
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Abdel, Rahim Ma'en Ahmad. „Gene silencing in cancer cells using siRNA : genetic and functional studies“. Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/218.
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Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.
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Tezcan, Okan. „Metastatic Behaviour Of Doxorubicin Resistant Mcf-7 Breast Cancer Cells After Vimentin Silencing“. Master's thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615553/index.pdf.
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Chemotherapy is one of the common treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). MDR is caused by the activity of diverse ATP binding cassette (ABC) transporters that pump drugs out of the cells. There are several drugs which have been used in treatment of cancer. One of them is doxorubicin that intercalates and inhibits DNA replication. However, doxorubicin has been found to cause development of MDR in tumors. It has been reported that there is a correlation between multidrug resistance and invasiveness of cancer cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness. In this study, MCF-7 cell line (MCF-7/S), which is a model cell line for human mammary carcinoma, and doxorubicin resistant MCF-7 cell line (MCF-7/Dox) were used. The resistant cell line was previously obtained by stepwise selection in our laboratory. The main purpose of this study was to investigate changes of metastatic behaviour in MCF-7/Dox cell line, after transient silencing of vimentin gene by siRNA. In conclusion, down-regulation of vimentin gene expression in MCF-7/Dox cell lines was expected to change the characteristics in migration and invasiveness shown by migration and invasion assays.
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Labrèche, Cédrik. „The Role of Periostin in ErbB2-Driven Mammary Tumorigenesis and its Gene Regulation in ErbB2+ Cancer Cells“. Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42753.
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Breast cancer is a highly heterogeneous disease with multiple drivers and a complex regulatory network. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. More specifically, Postn has been shown to be involved in various processes of tumor progression such as angiogenesis, cell survival, invasion, and metastasis. A high Postn level in breast cancer has been corelated with a more aggressive phenotype. Despite extensive research, it remains unclear what Postn is doing to the cancer environment and how cancer cells regulate Postn. Here, we assessed the role and regulation mechanisms of Postn in ErbB2-mediated tumorigenesis. By crossing Postn deficient animals into the oncogenic NeuNDL model of ErbB2-positive breast cancer, we have shown that Postn deletion delays tumor onset and increases overall survival by affecting proliferation and apoptosis. These tumors also showed a decrease in collagen deposition which is the proposed mechanism for its effect in vivo. Using isolated cancer cells from the Postn deficient background we assessed re-expression of Postn which had no effect on in vitro tumorigenesis processes or in vivo subcutaneous growth in immunodeficient mice. Furthermore, we established an in vitro model to study the regulation of Postn using a bovine pituitary gland derived extract as a natural repressor of Postn. Using mass spectrometry and RNA sequencing, we identified potential regulators of Postn gene expression. We also showed a cross regulation between FGFR, TGFβ and PI3K/AKT pathways to regulate Postn expression. In ErbB2-mediated murine breast cancer cells, we found that TGFβ can induce Postn expression in a SMAD-independent manner while bFGF can repress Postn expression through a PKC-dependent pathway. Postn induction and repression by TGFβ and bFGF respectively, are both dependent on PI3K/AKT signaling. Overall, these results suggest a cancer-driving function for Postn and reveal a novel mechanism for regulating Postn expression.
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Wood, Timothy Paul. „Identification of key mechanism in the cytotoxic effect of two novel anti-cancer compounds on breast cancer cells“. Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/24532.
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Organometallic chemotherapeutic agents, many of which target DNA, have been shown to be effective in the treatment of cancer. With that said though, these compounds have a number of side affects such as nephrotoxicity. Two novel compounds, Ferrocene [ferrocenoyltrichloroacetone] and Rhodium-ferrocene [(1.5 cyclooctadiene)(1-ferrocenyl- 4,4,4-trichloro-1,3-butanedionate], synthesised by the research group of J Swarts (University of the Free State) were evaluated to determine their mechanism of action and their potential use as novel therapeutic agents. It is hypothesized, by merit of their chemical structures, that these compounds’ anti-cancer activity is due to their interaction with DNA. Both drugs were evaluated from a cellular to a molecular level, in vitro, to validate this hypothesis. Linearised DNA was exposed to both drugs and digested with a variety of restriction enzymes. It was found that the compounds bind to the PstI restriction site; thereby inhibiting the enzyme’s restriction activity. From this point it was necessary to show that the compounds are able to interact with DNA in a cellular system. By exposing a transformed breast epithelial cell line (MCF-12A) and a cancerous breast epithelial cell line (MCF-7) to the compounds, for various times, followed by flow cytometric analyses, it was found that both affect progression through the cell cycle. Cells accumulated at various phases of the cell cycle, as a result of checkpoint gene activation. Further flow cytometric analyses showed that both drugs induce necrosis in MCF-7 cells. The “normal” cell line however did not show this response as it is believed that cell cycle arrest and repair mechanisms were initiated, which would delay cell death. Gene expression analyses were performed by reverse transcriptase real-time PCR in which panels of cell cycle related genes as well as DNA damage associated genes were probed in two separate array formats. These studies revealed that a number of DNA damage and repair genes are activated; specifically those associated with excision repair and free-radical induced DNA damage. Members of the RAD family as well as the genes GADD45A, XPC and OGG1 were found to be upregulated as a result of Ferrocene treatment. This could be expected as it was shown that ferrocene binds to DNA, and it logically then follows that this would lead to excision repair being attempted by the cell. Similar gene expression patterns were found following Rhodium-ferrocene treatment with the up-regulation of genes such as OGG1, ATM and GADD45G, albeit to a lesser extent. It is hypothesised that the larger molecule may not interact as effectively with DNA, due to steric hinderance. Arrest mechanisms, for both drugs, were more pronounced in the “normal” cell line and it is believed that this is due to the fact that many of these genes have been inactivated in the cancerous cell line. We have shown, on multiple levels, that both compounds’ therapeutic action is as a result of their interaction with the cell’s DNA. This interaction leads to cell death in both the transformed and the cancerous cell line. In order to clarify these mechanisms it is suggested that proteomic and metabolomic studies should be performed.Dissertation (MSc)--University of Pretoria, 2012.
Genetics
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Sancho, Medina Mònica. „Role of linker Histone H1 variants in cell proliferation, Chromatin Structure and Gene expression in breast cancer cells“. Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7118.
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At least eleven histone H1 variants exist in mammalian somatic cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition of playing a structural role, H1 seems to be involved in the activation and repression of gene expression. It is not well known whether the different variants have specific roles or regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated by changes of expression of other variants. A different, reduced subset of genes is altered in each H1 knock-down. Interestingly, H1.2 depletion represses expression of a number of cell cycle genes. This is concomitant with a G1 arrest phenotype observed in this cell line. In addition, H1.2 depletion caused decreased global nucleosome spacing. These effects are specific of H1.2 depletion as they are not complemented by overexpression of other variants and they do not occur in knock-downs for the other variants. Moreover, H1.4 depletion caused cell death in T47D, being the first report of the essentiality of an H1 variant for survival in a human cell type. In addition to this, we have also investigated specificities of H1 subtypes location in particular promoters of interest in our laboratory, as well as specific interactions with other factors by generating HA-tagged H1 variant expressing cell lines.Al menos once variantes de la histona H1 han sido identificadas en mamíferos, todas ellas se unen al ADN entre nucleosomas contribuyendo así, a la estabilización de la partícula nucleosómica y a la compactación de la cromatina en estructuras de alto orden. Además de jugar un papel estructural, H1 parece estar implicada en la activación y represión de la expresión génica. Se desconoce si las diferentes variantes de H1 tienen funciones específicas o regulan promotores específicos. Con el objetivo de investigar esta hipótesis se han generado líneas celulares que inhiben de forma inducible, mediante la tecnología de ARN interferente, la expresión de cada una de las variantes de forma específica. La inhibición de cada una de las variantes no es compensada por cambios en la expresión del resto de subtipos. Distintos grupos de genes resultan alterados con la depleción de cada una de las variantes de H1. La inhibición de H1.2 reprime la expresión de una serie de genes de ciclo celular, correlacionando con un fenotipo de arresto celular en fase G1 observado en esta línea. Además, la inhibición de H1.2 causa una disminución global del espaciamiento entre nucleosomas. Todos estos efectos parecen ser específicos para la falta de H1.2 ya que no son complementados por la sobreexpresión de otras variantes. Por otro lado, la inhibición de H1.4 causa muerte celular en T47D. Ésta es la primera vez que se describe que una variante de H1 es esencial para la supervivencia de una línea celular humana.
En un segundo plano, se han construido líneas celulares con expresión de las variantes de H1 fusionadas al péptido HA, con el objetivo de estudiar la especificidad de su localización en promotores de interés para el grupo, así como interacciones específicas con otros factores celulares.
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Kim, Ju Young. „Transcriptional regulation of glycosyltransferase genes in MCF-7 human breast cancer cell line following drug treatment“. Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29866.
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Bioinformatics is a subfield in computational science that is principally focused on developing methods and performing data analytics in the areas of proteomics and genomics. In this thesis I draw a link between proteomics and genomics by focusing on the regulation patterns of glycosyltransferase (GT) genes in breast cancer cell line following the treatment with a large set of Food and Drug Administration (FDA) approved drugs. This is based on the understanding that aberrant glycosylation in breast cancer tumours stem from altered GT gene expression. A major goal of genomic research is the identification of genes that have been differentially expressed under abnormal conditions. A gene expression profile provides a snapshot of the transcriptional level of a cell. A comparative gene expression profile between a diseased and normal-state can be used to map out the regulatory mechanisms of disease. In this thesis, the results of Microarray experiments on MCF-7 human breast cancer cell-lines are analysed using statistical and computational tools to identify differentially expressed genes. Here a bioinformatics analysis of the regulation of GT gene expressions was performed to identify a set of glycosylation related genes with the aim of making an inference about their biological functions. A set of raw gene expression profiles from MCF-7 human breast cancer cell-line treated with different therapeutic drugs were obtained from the Connectivity Map (CMap) database. Initially 7,000 gene expression profiles were used and these were treated by 1,309 different FDA-approved drugs. The number of genes initially was counted up to 22,000. Using the Bioconductor open source software in R statistical programming environment a statistical differential expression analysis followed by several data filtering and pre-processing steps were performed to identify up and down regulated GT genes using. Using non-parametric rank sum meta-analysis three cancer drugs and two non-cancer drugs were identified as effective agents able to control the transcriptional regulatory state of GT genes. The study concluded by employing co-expression gene module analysis using the Weighted Gene Co-Expression Network Analysis (WGCNA) package on each of the cancer and non-cancer drug treatments. The gene modules discovered from the analysis were used to perform gene ontology enrichment analysis to identify the biological functions where they were significantly enriched in. The co-expression modules where GT genes have been down regulated by the drugs, were involved in processes such as Wnt signalling and cell surface pattern recognition receptor signalling important for cancer development. Immune response and apoptotic processes in the cell were identified from co-expression modules where GT genes were up regulated. This key finding that the GT gene expressions are markers for treatment analysis points to their use in drug development studies. The second more direct finding is that non-breast cancer specific FDA-approved drugs may have a role in treating breast cancer and may be the subject of future drug repurposing strategies.
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Husbeck, Bryan. „Changes in gene expression induced by thioredoxin-1 in MCF-7 human breast cancer cells“. Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/280113.
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Thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human cancers. Elevated levels of Trx-1 in tumors is associated with increased cell proliferation, decreased apoptosis, and decreased patient survival. However, the mechanism(s) for the growth stimulating and anti-apoptosis effects of Trx-1 are unknown. We used DNA microarray technology to identify genes whose expression was altered in MCF-7 breast cancer cells stably transfected with wild-type Trx-1 (MCF-7/Trx 9) or a redox inactive mutant Trx-1 (MCF-7/SerB 4) compared to empty-vector transfected cells (MCF-7/neo). The expression of cytochrome P450 1B1 (CYP1B1) mRNA and protein is increased by Trx-1 transfection of MCF-7 human breast cancer cells and decreased by a redox inactive mutant Trx-1. CYP1B1 is a tumor specific CYP which converts 17β-estradiol (E₂) to the carcinogenic 4-hydroxyestradiol (4-OHE₂). The expression of peroxiredoxin 1 (PRDX1) mRNA is increased as a result of Trx-1 overexpression in MCF-7 cells. The peroxiredoxins belong to a conserved family of antioxidant proteins that use thiol groups as reducing equivalents to scavenge oxidants. Transfection of mouse WEHI7.2 thymoma cells with human PRDX1 protects cells from apoptosis induced by H₂O₂. Spermine/spermidine N'-acetyltransferase (SSAT) mRNA expression and enzyme activity is decreased by Trx-1 transfection of MCF-7 human breast cancer cells. SSAT is an important enzyme in the polyamine catabolic pathway. The inhibition of SSAT enzyme activity is associated with decreased putrescine levels in the Trx-1 transfected cells. Therefore, it appears as if the modification of cellular redox signaling brought about by the overexpression of Trx-1 in breast cancer cells induces changes in gene expression that contribute to the transformed phenotype. Trx-1 redirects estrogen metabolism in a more toxic pathway due to the induction of CYP1B1, provides resistance to apoptosis induced by reactive oxygen species via the upregulation of PRDX1, and alters polyamine metabolism by inhibiting the expression of SSAT.
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Bellucci, Luca. „The role of histone variant H2A. Z in the regulation of gene expression in breast cancer cells“. Toulouse 3, 2013. http://thesesups.ups-tlse.fr/1939/.
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Pendant mes trois ans de thèse, j'ai étudié les mécanismes de régulation génique impliqués dans le cancer du sein. Je me suis intéressé en particulier à la régulation épigénétique du gène p21 dans les cellules cancéreuses mammaires hormono-indépendantes, ERa-négatives (MDA-MB231). Nous avons démontré que l'activation du gène p21 dans ces cellules était p53 indépendante, stimulée en utilisant des inhibiteurs des histones deacétylases et contrôlée par l'acétylation du variant d'histone H2A. Z. Mes études ont permis la rédaction et publication d'un article (Bellucci et al. , 2013 [1]). Selon les cellules utilisées, l'expression du gène p21 peut toutefois être p53 dépendante ou indépendante et le mécanisme d'activation du gène p21, via les HDACi, reste peu compris. Par ailleurs, il a été démontré, dans autres systèmes cellulaires, que la régulation de p21 dépendait de la localisation du variant d'histone H2A. Z. Nous avons montré que dans les cellules MDA-MB231 (p53- négatives), H2A. Z est lié fortement au site d'initiation de la transcription du gène p21. Un traitement à la Trichostatin A (TSA), un inhibiteur des histone-deacétylases, augmente l'acétylation de H2A. Z favorisant ainsi l'expression de p21. A l'opposé, une cellule déplétée en H2A. Z ne répond plus aux HDACi. Nous avons démontré que la présence du variant d'histone H2A. Z dans sa forme acétylé était importante pour l'expression de p21. En utilisant comme gène modèle celui de la Cycline D1, nous avons montré que l'acétylation de H2A. Z joue un rôle fondamental dans la régulation de la transcription en présence et en absence du récepteur aux œstrogènes. De plus, nous avons identifié des enzymes impliqués dans l'acétylation de H2A. Z (Tip60) et dans le remodelage de la chromatine (Tip48), nous permettant de proposer un modèle pour l'activation de la transcription de ce gèneDuring my PhD, I studied the mechanisms of regulation of gene expression in breast cancer. In particular, I was interested in the epigenetic regulation of p21 gene expression in an estrogen receptor negative breast cancer cells (MDA-MB231 cells). We found that in these cells a TSA treatment stimulated p21 expression and increased acetylation of H2A. Z present at the p21 promoter. H2A. Z was strongly associated with the transcription start site of p21. Moreover, depleting the cellular pool of H2A. Z compromised p21 activation and response to HDACi. Acetylation of H2A. Z rather than its association of regulatory element per se was important for p21 expression. My studies led to a publication (Bellucci et al. , 2013 [1]) which shows that p21 gene activation in MDA-MB231 cells is p53-independent and controlled by the H2A. Z histone variant and its acetylation. Normally, activation of p21 expression can be p53-independent or dependent, according to the cell system used. But the mechanism behind p21 activation, via HDACi, remains poorly understood. Moreover, p21 regulation depends on the binding of the histone variant H2A. Z. Using the Cyclin D1 gene as a model, I also participated in a project, which shows how, in this case too, H2A. Z acetylation is critical for gene regulation in estrogen receptor positive and negative breast cancer cells. We identified the enzymes involved in H2A. Z acetylation (Tip60) and in chromatin remodeling (Tip48), to propose a model for transcription activation of this gene
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Hunter, Rebecca Stephanie. „Inhibition of ErbB2 and Thymidylate Synthase by a Multi-Targeted Small-Interfering RNA in Human Breast Cancer Cell Lines“. Yale University, 2008. http://ymtdl.med.yale.edu/theses/available/etd-08092007-140112/.
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The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.
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Higgins, Kelly Jean. „Regulation of vascular endothelial growth factor receptor-2 in pancreatic and breast cancer cells by Sp proteins“. Texas A&M University, 2003. http://hdl.handle.net/1969.1/6010.
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Vascular endothelial growth factor receptor-2 (VEGFR2) is a key
angiogenic factor, and angiogenesis is an important physiological process
associated with neovascularization, growth, and metastasis of many different
tumors. The mechanism of VEGFR2 gene expression was investigated in
MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a
series of VEGFR2 promoter deletion/mutated constructs, and the results
indicated that the GC-rich âÂÂ60 to âÂÂ37 region of the promoter was essential for
VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp
proteins are expressed and bind to the proximal GC-rich region of the VEGFR2
promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3,
and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic
cancer cells.
VEGFR2 gene expression was also investigated in ZR-75 and MCF-7
breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased
VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced
by E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSA
and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in
ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter.
RNA interference was used to determine the relative contributions of Sp proteins
on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly,
in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and
ERa/Sp4 but not ERa/Sp1.
In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were
decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich
region important for E2-mediated upregulation in ZR-75 cells was responsible for
E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells.
EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are
expressed in MCF-7 cells and bind to the proximal GC-rich region of the
VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4
are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and
ERa/Sp protein-promoter interactions are accompanied by recruitment of the
corepressor SMRT using the ChIP assay.
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D’Anello, Laura <1980>. „Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells“. Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3368/.
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Gu-Trantien, Chunyan. „Gene expression profiling of CD4+ T cells infiltrating human breast carcinomas identified CXCL13-producing T follicular helper cells associated with tertiary lymphoid structures and better patient outcome“. Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209474.
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Over the past decade, studies using murine models have led to the demonstration that CD4+ T helper (Th) cells play a critical role in the control of cancer progression. Additional support for their importance comes from the growing body of recent clinical/translational research data demonstrating the importance of tumor-infiltrating T and B lymphocytes in long-term patient survival for various types of cancer, including breast cancer (BC). As the key population coordinating adaptive immune responses, the role(s) played by individual Th subsets in cancer immunity remains largely controversial. The Th1 subset has uniquely been shown to have a clear anti-tumor effect, guiding CD8+ cytotoxic T cells-mediated direct tumor cell lysis through IFN-γ secretion. Although the negative regulatory role played by Treg cells has been extensively studied in cancer, its prognostic value along with that of Th2 and Th17 cells have not been clearly demonstrated in patients. T follicular helper (Tfh) cells, a recently characterized Th subset that plays a primary role in the generation of B cell memory in secondary lymphoid organs, have not been previously described infiltrating solid tumors. The principal objective of this thesis was to perform an in-depth characterization of tumor-infiltrating CD4+ T cells (TIL) and Th subsets in human BC, where very little is currently known.
Using whole genome microarrays, we analyzed the gene expression profiles of TIL relative to their counterparts from the axillary lymph nodes and peripheral blood. Applying a novel approach, we compared TIL profiles with public microarray data for Th subsets, demonstrating: 1) the presence of all major Th subsets (Th1, Th2, Th17, Treg as well as Tfh) in the TIL, 2) the TIL are effector memory rather than central memory cells, 3) the TIL are concomitantly activated and suppressed and 4) TIL from tumors with extensive lymphoid infiltrates are more activated/less suppressed in the TCR/CD3 signaling pathway, producing higher levels and a wider panel of Th cytokines than TIL from minimally-infiltrated tumors.
We also performed in vitro experiments to study tumor microenvironment effects on TIL by treating normal CD4+ T cells from healthy donor blood with primary tumor supernatants (SN). Tumor SN largely reproduces the TIL profile in normal Th cells, totally suppressing their activation and inhibiting their cytokine production. Intriguingly, the highly restricted number of cytokines induced by tumor SN included several tumor-promoting factors, such as IL-8 and TNF. SN from an extensively-infiltrated tumor was found to be less immune-suppressive than SN from minimally-infiltrated tumors. In line with this, TIL from minimally-infiltrated tumors are closer to SN-treated (suppressed) activated donor cells whereas TIL from extensively-infiltrated tumors are more similar to activated cells without SN treatment.
These results led us to further investigate the observed differences between TIL from extensive and minimally-infiltrated tumors. Genes characterizing Th1 and Tfh cells were enriched in the extensively-infiltrated tumors. PD-1hiCD200hi Tfh cells were specifically detected in extensively-infiltrated tumors by flow cytometry and these cells were determined to be the major source of the chemokine CXCL13. Immunohistochemical analysis demonstrated highly-organized tertiary lymphoid structures (TLS) within the tumor, containing a CD4+/CD8+ T cell zone and a B cell zone with reactive germinal centers where Tfh cells and follicular dendritic cells (FDC) are resident. Their presence suggests the origin of an effective memory anti-tumor immune response.
Finally, we generated Tfh- and Th1-specific gene signatures reflecting differences between extensive and minimal TIL and tested their prognostic value in large-patient-scale public data sets. Our Tfh signature predicts better 10-year disease-free survival for all BC subtypes, outperforming the Th1 signature, suggesting that Tfh cells play a more central role than Th1 cells in anti-tumor immunity. CXCL13 is the determinant gene of our Tfh signature, showing particularly strong prognostic power for the HER2+ subtype. Additionally, these signatures also predict a better response to neoadjuvant chemotherapy.
This thesis research has demonstrated that a previously undetected Th subset, Tfh cells, infiltrates solid tumors and shown that their presence signals enhanced anti-tumor immunity.
Durant cette dernière décennie, des travaux menés dans des modèles murins ont permis de mettre en évidence le rôle crucial joué par les lymphocytes T auxiliaires CD4+ (Th) dans le contrôle de la progression des cancers. De plus, de nombreuses études cliniques et/ou translationnelles récentes corroborent ces observations en montrant une corrélation entre l’importance de l’infiltration intra-tumorale par les lymphocytes T et B et la survie à long terme des patients atteints de différents types de cancer, dont le cancer du sein. En tant que chefs d’orchestre de la réponse immune adaptative, les rôles spécifiques des sous-populations des cellules Th restent controversés. Les Th1 sont la seule population exerçant une claire réponse anti-tumorale, qui est liée à la sécrétion d’IFN-γ, une cytokine primordiale à l’action des lymphocytes T cytotoxiques CD8+. Bien que le rôle néfaste des T régulateurs (Treg) a été largement étudié dans le cancer, leur implication pronostique ainsi que celle des Th2 et Th17 n’ont pas encore été clairement démontrées. La présence d’une sous-population de CD4, les T auxiliaires folliculaires (Tfh), cellules clés dans la différenciation des lymphocytes B mémoires au sein des organes lymphoïdes secondaires, n’a jamais été décrite dans les cancers solides. Le but principal de ce travail est de caractériser les sous-populations des lymphocytes T CD4+ infiltrant la tumeur (TIL) en prenant comme modèle le cancer du sein humain. A l’heure actuelle, il existe très peu de données sur les TIL CD4 dans ce type de cancer.
Nous avons d’abord établi le profil génique des TIL en les comparant avec ceux provenant des ganglions axillaires ou du sang périphérique. En appliquant une nouvelle approche, nous avons comparé les profils des TIL avec les données publiques de sous-populations de Th et démontré que :1) toutes les sous-populations de cellules Th (Th1, Th2, Th17, Treg et Tfh) infiltrent la tumeur, 2) les TIL ont un phénotype plus proche de celui des cellules mémoires effectrices que des cellules mémoires centrales, 3) les TIL sont simultanément activés et supprimés et 4) les TIL provenant des tumeurs massivement infiltrées («extensives») par des lymphocytes sont mieux activés et moins supprimés que les TIL des tumeurs peu infiltrées («minimales») dans la voie de signalisation TCR et produisent des cytokines d’une quantité plus élevée et d’une répertoire plus large.
Nous avons également effectué des expériences in vitro pour étudier l’effet de l’environnement tumoral sur les TIL en traitant des CD4 normaux (provenant des donneuses saines) par le surnageant (SN) extrait des tumeurs fraiches. Le SN tumoral induit un profil génique proche de celui des TIL en inhibant l’activation et la production de cytokines de ces cellules stimulées. Curieusement, parmi le peu de cytokines induites par le SN tumoral, des facteurs pro-tumoraux comme IL-8 et TNF sont détectés. Le surnageant provenant d’une tumeur «extensive» est moins immunosuppresseur que ceux des tumeurs «minimales». Conformément, les TIL provenant des tumeurs «minimales» ont un profil génique proche des cellules normales activées et traitées (supprimées) par le SN tumoral tandis que les TIL des tumeurs «extensives» ressemblent aux cellules activées non traitées.
Ces résultats nous avaient guidés à investiguer plus profondément les différences observées entre les TIL des tumeurs «extensives» et «minimales». Les gènes caractéristiques des Th1 et Tfh sont enrichis dans les tumeurs «extensives». Les cellules Tfh PD1hiCD200hi sont spécifiquement détectées par cytométrie de flux dans les tumeurs «extensives» et sont identifiées comme les producteurs principaux de la chimiokine CXCL13. L’examen par immunohistochimie a permis de détecter des structures lymphoïdes tertiaires (TLS) dans la tumeur, composées d’une zone T (CD4 et CD8) et d’une zone B au sein de laquelle se trouve parfois un centre germinatif actif contenant des Tfh et des cellules dendritiques folliculaires (FDC). La présence de ces structures suggère l’origine d’une réponse immune mémoire anti-tumorale.
Finalement, nous avons établi des signatures géniques spécifiques aux Tfh et Th1 et recherché leur impact pronostique dans deux bases de données publiques à grande échelle. Notre signature Tfh est positivement corrélée avec la survie à 10 ans des patientes de tous les sous-types de cancer du sein, et est plus performante que la signature Th1. Ceci suggère que les Tfh pourraient jouer un rôle plus crucial que les Th1 dans la réponse immune anti-tumorale. CXCL13 est le gène déterminant de notre signature Tfh et son expression est fortement associée à une meilleure survie chez les patientes du sous-type HER2+. De plus, ces signatures prévoient également une meilleure réponse à la chimiothérapie néoadjuvante (préopératoire).
Cette étude a démontré qu’une nouvelle sous-population de CD4, les Tfh, infiltre la tumeur solide et leur présence indique l’existence d’une immunité anti-tumorale renforcée.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
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Stone, Andrew. „Identification of genes associated with the maintenance of the tamoxifen resistant breast cancer cell phenotype following tamoxifen withdrawal“. Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55814/.
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Despite the benefit tamoxifen has provided for millions of breast cancer patients worldwide, almost all patients with metastatic disease and as many as 40 of patients receiving adjuvant tamoxifen treatment will acquire resistance to the drugs inhibitory effect on breast cancer cell growth. Previous studies in the Tenovus Centre have demonstrated that the development of anti-oestrogen resistance in vitro is associated with aberrant growth factor signaling which facilitates a more aggressive cell phenotype. The aim of the present study was to determine whether the undesirable characteristics of tamoxifen resistant cells were maintained following withdrawal from the drug. Interestingly, the accelerated rate at which resistant cells proliferated was sustained following a 6 month withdrawal period despite decreased expression of epidermal growth- factor receptor and reduced sensitivity to gefitinib. Following the assessment of long-term tamoxifen exposure on classically regulated oestrogen gene targets progesterone receptor and trefoil factor 1, it was apparent that the genes were no longer inducible by oestradiol following the acquisition of resistance. In contrast, when cells were co-treated with a demethylation agent in combination with oestradiol, genes were once again responsive to oestrogen stimulation, providing proof of principle that long-term tamoxifen exposure can silence oestrogen regulated gene expression through promoter hyper- methylation. Importantly, this combination treatment was shown to significantly reduce cell growth, inferring that a proportion of the genes that were reactivated by this treatment were associated with a tumour suppressive function. Using microarray technology, methylight analysis and polymerase chain reaction validation, several genes with tumour-suppressive ontology were identified as being silenced by promoter hypermethylation in tamoxifen-1 withdrawn tamoxifen-resistant cells, including p53 gene target, prostate differentiation factor, and inhibitor of Ras signaling, Ras protein activator-like 1. It is therefore proposed that anti-hormone induced epigenetic modification of tumour-suppressor genes, alongside aberrant growth factor signaling, can promote resistant cell survival and progression.
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Singh, Anjana. „Development of a reporter gene assay to determine estrogen receptor activity in purified primary breast cancer cells“. Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418052.
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Man, Yicun [Verfasser]. „On the functional relevance of the gene DEP domain containing 1 in breast cancer cells / Yicun Man“. Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/1035405989/34.
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Montanez-Wiscovich, Marjorie E. „Discerning the Role of LMO4 as a Global Modulator of G2/M Cell Cycle Progression and Centrosome Cycle in Breast Cancer Cells“. Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1259899890.
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Jeffy, Brandon David. „Molecular interactions between endogenous and exogenous factors: Regulation of BRCA-1 tumor suppressor gene expression in breast cancer cells“. Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280421.
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This dissertation focuses on the central hypothesis that in breast cancer cells containing the estrogen receptor-α (ER-α+) and wild-type p53, the BRCA-1 tumor suppressor gene is positively regulated by the steroid hormone estrogen and negatively regulated by Aromatic Hydrocarbon Receptor (AhR) ligands which damage DNA. In this dissertation, we demonstrate that BRCA-1 promoter activity is reduced by the DNA damaging agent Benzo[a]pyrene in breast cancer cells containing both a functional estrogen receptor and p53 pathway. In addition, our data suggests that exposure of MCF-7 breast cancer cells to estrogen stimulates transcription from the BRCA-1 5 ' flanking region, and this increase in transcription is paralleled by an increase in estrogen receptor-alpha interaction at the BRCA-1 promoter between -46 → -14 upstream of exon 1b. We report that in both untreated and estrogen-treated M CF-7 cells, a transcriptional complex, which we have termed an "Estrogen Responsive Unit" (ERU), containing AP-1, Sp1, and CREB family members, forms at the same -46 → -14 region which binds ER-α. Moreover, we show that wild-type p53 is required for estrogen induction of BRCA-1 and overexpression of a dominant-negative mutant variant of p53 can prevent this induction. Finally, we show that overexpression of wild-type p53 is able to disrupt the estrogen receptor interaction with the BRCA-1 ERU under both basal and estrogen-induced conditions while mutant p53 is only able to disrupt this interaction when estrogen is present. Taken together, these data suggest that loss of function of either the estrogen receptor-α or p53 signaling pathways may result in an inability for BRCA-1 regulation to occur and may in turn be a risk factor in the etiology of sporadic breast cancer.
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Ghatge, Radhika P. „Medroxyprogesterone acetate : a dual function hormone that is both a progestin and an androgen in human breast cancer cells /“. Connect to full text via ProQuest. IP filtered, 2005.
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Mardilovich, Katerina. „Insulin Receptor Substrate-2 (IRS-2): A Novel Hypoxia-Responsive Gene in Breast Cancer: A Dissertation“. eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/533.
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Breast cancer is the most common malignancy among women in the U.S. While many successful treatments exist for primary breast cancer, very few are available for patients with metastatic disease. The purpose of this study was to understand the role of Insulin Receptor Subtrate-2 (IRS-2) in breast cancer metastasis. IRS-2 belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, many of which have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with cancer cell invasion, glycolysis and tumor metastasis. In the current work, we identified IRS-2 as a novel hypoxia-responsive gene in breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have shown that IRS-2 upregulation in response to hypoxia promotes Akt signaling and tumor cell viability and invasion. We identified a cell context-dependent role for Hypoxia Inducible Factor (HIF) in the regulation of IRS-2 expression in hypoxia, with HIF-2 playing a more dominant role than HIF-1. We also demonstrate that binding of Snail, a regulator of the EMT, to the IRS-2 promoter keeps the chromatin in an open conformation that is permissive for HIF-dependent transcription of IRS-2 in hypoxia. IRS-2 is not upregulated by hypoxia in well-differentiated epithelial-like carcinoma cells that do not express Snail, implicating IRS-2 gene expression as part of the EMT programming. In summary, we have identified an endogenous mechanism by which cancer cells can shift the balance of IRS-1 and IRS-2 to favor IRS-2 expression and function, which promotes survival, invasion, and ultimately metastasis. Understanding the mechanism of IRS-2 regulation by hypoxia may reveal new therapeutic targets for metastatic breast cancer.
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Richter, Suzanne C. „New approaches to molecular diagnostics, identification of differentially expressed genes in breast cancer cell lines using cDNA aray hybridization and sequence analysis“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0009/MQ40773.pdf.
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Yilmaz, Selis. „Antioxidant And Cytotoxic Properties Of Salvia Absconditiflora And Effects On Cyp1a1, Cyp1b1 Gene Expressions In Breast Cancer Cell Lines“. Master's thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615663/index.pdf.
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Salvia genus is a widely cultivated genus and used in medicine for various purposes as having antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim was to investigate phenolic composition of Salvia absconditiflora and understand the possible effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of total phenolic content. Presence of important phenolic acids and flavonoids such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis.
Cytotoxicity of Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were investigated through XTT and TBE assays both dose and time dependent manner. Cell proliferation was inhibited 50 % by different IC50 values calculated in different assays and different time intervals. This suggests that two breast cancer cell lines response in a different way to cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were investigated with qRT-PCR. CYP1A1 and CYP1B1 were upregulated in MCF-7 but down-regulated in MDA-MB-231 cells in response to Salvia absconditiflora treatment.
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Albuquerque, Andreia de, Sepp Kaul, Georg Breier, Petra Krabisch und Nikos Fersis. „Multimarker Analysis of Circulating Tumor Cells in Peripheral Blood of Metastatic Breast Cancer Patients: A Step Forward in Personalized Medicine“. Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136627.
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Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoringZiel: Entwicklung eines immunomagnetischen Verfahrens zur Isolierung zirkulierender Tumorzellen (CTCs) in Kombination mit einer molekularen Multimarkeranalyse für die hochspezifische Identifizierung maligner Zellen. Patientinnen und Methoden: Peripheres Blut (PB) von 32 Patientinnen mit metastasiertem Mammakarzinom und von 42 gesunden Kontrollen wurde für die immunomagnetische Tumorzellanreicherung mit den Antikörpern BM7 und VU1D9 genutzt. Eine Real-Time Reverse Transkription Polymerase-Kettenreaktion (RT-PCR)-Methodik mit den Markern KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 und ERBB2 wurde für den CTC-Nachweis und die Tumorzellcharakterisierung entwickelt. Ergebnisse: Für die einzelnen Marker wurden die folgenden Positivitätsraten ermittelt: 46,9% für KRT19, 25,0% für SCGB2A2, 28,1% für MUC1, 28,1% für EPCAM, 21,9% für BIRC5 und 15,6% für ERBB2. Nach der Bestimmung individualisierter Cut-off-Werte ergab sich für den kombinierten Multimarkernachweis eine Sensitivität und Spezifität von 56,3% bzw. 100%. Bemerkenswert war der Befund, dass 27,0% der HER2-tumornegativen Patientinnen ERBB2-mRNA-positive CTCs aufwiesen. Schlussfolgerung: Die hier beschriebene Methodik bestimmt CTCs mit hoher Spezifität. Die molekulare Multimarkeranalyse liefert wertvolle Real-Time-Informationen für personalisierte Behandlungsmodalitäten
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Albuquerque, Andreia de, Sepp Kaul, Georg Breier, Petra Krabisch und Nikos Fersis. „Multimarker Analysis of Circulating Tumor Cells in Peripheral Blood of Metastatic Breast Cancer Patients: A Step Forward in Personalized Medicine“. Karger, 2012. https://tud.qucosa.de/id/qucosa%3A27718.
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Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoring.Ziel: Entwicklung eines immunomagnetischen Verfahrens zur Isolierung zirkulierender Tumorzellen (CTCs) in Kombination mit einer molekularen Multimarkeranalyse für die hochspezifische Identifizierung maligner Zellen. Patientinnen und Methoden: Peripheres Blut (PB) von 32 Patientinnen mit metastasiertem Mammakarzinom und von 42 gesunden Kontrollen wurde für die immunomagnetische Tumorzellanreicherung mit den Antikörpern BM7 und VU1D9 genutzt. Eine Real-Time Reverse Transkription Polymerase-Kettenreaktion (RT-PCR)-Methodik mit den Markern KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 und ERBB2 wurde für den CTC-Nachweis und die Tumorzellcharakterisierung entwickelt. Ergebnisse: Für die einzelnen Marker wurden die folgenden Positivitätsraten ermittelt: 46,9% für KRT19, 25,0% für SCGB2A2, 28,1% für MUC1, 28,1% für EPCAM, 21,9% für BIRC5 und 15,6% für ERBB2. Nach der Bestimmung individualisierter Cut-off-Werte ergab sich für den kombinierten Multimarkernachweis eine Sensitivität und Spezifität von 56,3% bzw. 100%. Bemerkenswert war der Befund, dass 27,0% der HER2-tumornegativen Patientinnen ERBB2-mRNA-positive CTCs aufwiesen. Schlussfolgerung: Die hier beschriebene Methodik bestimmt CTCs mit hoher Spezifität. Die molekulare Multimarkeranalyse liefert wertvolle Real-Time-Informationen für personalisierte Behandlungsmodalitäten.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Silveira, Giórgia Gobbi da. „Relação entre o gene B-Cell-Specific Moloney Murine Leukemia Virus Integration Site 1 (BMI-1) e genes reguladores da recombinação homóloga em carcinomas ductais invasores da mama“. Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17143/tde-08082014-112232/.
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Bmi-1 é uma proteína do grupo Polycomb capaz de induzir atividade de telomerase, levando à imortalização de células epiteliais. As células, quando imortalizadas, se tronam mais susceptíveis a danos em dupla fita (double-strand breaks (DSB))e a recombinação homóloga é uma das duas vias de reparo dos DSBs. Dentre os genes reguladores da recombinação homóloga temos o BRCA-1, que está envolvido na resposta ao dano associado à proteína RAD51, que por sua vez se acumula rapidamente nos focos de dano ao DNA após a sinalização do H2AX, que têm se mostrado um excelente marcador de dano celular por se acumular rapidamente nos focos de lesão, desencadeando o processo de reparo. Topoisomerase III (TopoIII) remove intermediários da recombinação homóloga antes da segregação de cromossomos, prevenindo danos à estrutura do DNA celular. O papel das proteínas envolvidas na recombinação homóloga, em carcinomas ductais invasores positivos para o BMI-1, necessita ser investigado. Utilizando-se tissue microarrays contendo 239 casos de carcinomas ductais mamários primários, foi analisada a expressão imunoistoquímica de BMI-1, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1, H2AX, RAD51 e topoisomerase III. Positividade para o Bmi-1 foi encontrada em 66 casos (27.6%). A positividade imunoistoquímica do BMI-1 relacionou-se a RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) e TopoIII (p < 0,001). Concluindo, nossos resultados mostraram haver relação entre o BMI-1 e genes reguladores da HR, sugerindo que a positividade de BMI-1 pode ser um importante evento na recombinação homóloga em carcinomas ductais invasores da mama.Bmi-1 is a Polycomb group protein which is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells shown more susceptible to double-strand breaks (DSB) and the homologous recombination (HR) are one of DSB repair pathways. Among the regulatory genes in HR, there is BRCA1, involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX. H2AX has also been shown to be a good marker of DNA damage. Topoisomerase III (TopoIII) removes HR intermediates before the segregation of chromosomes, preventing damage to the structure of the cellular DNA. The role of proteins involved in HR, in breast carcinomas positive for BMI-1, remains to be investigated. The aim of this study was evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoisomerase III, RE, RP and HER-2 was analyzed by immunohistochemistry. We observe high expression of Bmi-1 in 66 cases (27.6%). Immunohistochemistry overexpression of BMI-1 was related to RE (p=0,004), RP (p<0,001), Ki-67 (p < 0,001), p53 (p=0,003), BRCA-1(p= 0,003), H2AX (p= 0,024) and TopoIII (p < 0,001). Our results showed a relation between the expression of BMI-1 and HR regulatory genes, suggesting that overexpression of Bmi-1 is an important event in breast cancer homologous recombination.
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Neil, Jason Robert. „TGF-ß promotes cancer progression through the xIAP:TAB₁:TAK₁:IKK axis in mammary epithelial cells /“. Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 117-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;