Analysis of the role of Hsp90 in colon cancer and cancer stem-like cell biology in vitro using a genetically paired cell line model
- Authors: Slater, Cindy
- Date: 2015-04-09
- Subjects: Heat shock proteins , Heat shock protein Hsp90 family , Colon (Anatomy) Cancer Treatment , Cell lines , Cancer stem-like cell , SW480 , SW620
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/480345 , vital:78433
- Description: Colon cancers are commonly associated with mutations or changes in signaling in the Wnt/β-catenin pathway. Cancer treatments, such as chemotherapy and radiation, are challenged by the limited methods of disease detection and the insufficient elimination of contributing factors, such as cancer stem-like cells (CSC), to metastatic disease. CSC are characterised by their ability to survive anchorage-independently and attribute to therapeutic resistance. To examine the biological changes associated with the progression of human colon cancers and the role of Hsp90 in cancer and CSC biology, SW480 and SW620 genetically paired (isogenic) colon cancer cell lines from the same patient were characterised for populations of putative CSC, tumoursphere (TS) forming ability, cell growth, behaviour and response to anti-cancer therapeutics. The SW480 cell line was established from a primary colon adenocarcinoma and the SW620 was established from a lymph node metastasis of the primary cancer one year later. To address the role of Hsp90 in colon cancer, the sensitivity of cells and TS were analysed in response to geldanamycin and novobiocin, and an isoform-specific approach to the targeting of Hsp90α was developed. Flow cytometric analysis of putative CSC by phenotype revealed variable proportions of cells bearing the CD44+/CD133+ surface protein marker, widely used in the identification of colon CSC, in SW480 and SW620 cells. The paired cell lines maintained similar proportions of putative CSC, identified by the expression of the ABCG2 protein (side population; 1 %) and through high aldehyde dehydrogenase activity (ALDEFLUOR; 6 %). SW480 cells demonstrated greater TS forming efficiency than SW620 cells (49.9 and 35.5 %, respectively) and observations of wound-healing showed SW480 cells to be more migratory than SW620 cells. No difference in response to Hsp90 inhibition was observed between paired cell lines, however SW480 TS resisted treatment with geldanamycin. This the first study to report a dose-dependent increase in TS growth in response to novobiocin inhibition of Hsp90, and to demonstrate that that the sensitivity of SW480 and SW620 TS to oxaliplatin, a common drug for the treatment of metastatic colon cancers, was enhanced by novobiocin, providing promise for the elimination of CSC with combined chemotherapeutics. We analysed the Wnt/β-catenin pathway in response to expression of short hairpin RNA (shRNA) against Hsp90α or a control non-targeting shRNA, under the control of a tetracycline-responsive promoter. Hsp90α knockdown contributed to a deregulated stress response, presenting with reduced Hsp27 and β-catenin protein, but corresponded to an increase in the association between Hsp90, β-catenin and Hsp27 in vitro. The reduction of Hsp90α did not influence sensitivity of the colon cancer cells to activators or inhibitors of the Wnt pathway, but rather correlated to reduced TS formation, cell adhesion and spreading, identifying potential therapeutic benefit to the controlled reduction of Hsp90α for the deregulation of colon cancer characteristics. Given that Hsp27 and β-catenin are both involved in cell adhesion, cytoskeletal dynamics and interact directly with each other, we propose a role for targeting Hsp90α in the regulation cell adherence indirectly via reductions in levels of β-catenin and Hsp27, rather than by modifying the transcriptional activity of β-catenin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015-04-09
- Authors: Slater, Cindy
- Date: 2015-04-09
- Subjects: Heat shock proteins , Heat shock protein Hsp90 family , Colon (Anatomy) Cancer Treatment , Cell lines , Cancer stem-like cell , SW480 , SW620
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/480345 , vital:78433
- Description: Colon cancers are commonly associated with mutations or changes in signaling in the Wnt/β-catenin pathway. Cancer treatments, such as chemotherapy and radiation, are challenged by the limited methods of disease detection and the insufficient elimination of contributing factors, such as cancer stem-like cells (CSC), to metastatic disease. CSC are characterised by their ability to survive anchorage-independently and attribute to therapeutic resistance. To examine the biological changes associated with the progression of human colon cancers and the role of Hsp90 in cancer and CSC biology, SW480 and SW620 genetically paired (isogenic) colon cancer cell lines from the same patient were characterised for populations of putative CSC, tumoursphere (TS) forming ability, cell growth, behaviour and response to anti-cancer therapeutics. The SW480 cell line was established from a primary colon adenocarcinoma and the SW620 was established from a lymph node metastasis of the primary cancer one year later. To address the role of Hsp90 in colon cancer, the sensitivity of cells and TS were analysed in response to geldanamycin and novobiocin, and an isoform-specific approach to the targeting of Hsp90α was developed. Flow cytometric analysis of putative CSC by phenotype revealed variable proportions of cells bearing the CD44+/CD133+ surface protein marker, widely used in the identification of colon CSC, in SW480 and SW620 cells. The paired cell lines maintained similar proportions of putative CSC, identified by the expression of the ABCG2 protein (side population; 1 %) and through high aldehyde dehydrogenase activity (ALDEFLUOR; 6 %). SW480 cells demonstrated greater TS forming efficiency than SW620 cells (49.9 and 35.5 %, respectively) and observations of wound-healing showed SW480 cells to be more migratory than SW620 cells. No difference in response to Hsp90 inhibition was observed between paired cell lines, however SW480 TS resisted treatment with geldanamycin. This the first study to report a dose-dependent increase in TS growth in response to novobiocin inhibition of Hsp90, and to demonstrate that that the sensitivity of SW480 and SW620 TS to oxaliplatin, a common drug for the treatment of metastatic colon cancers, was enhanced by novobiocin, providing promise for the elimination of CSC with combined chemotherapeutics. We analysed the Wnt/β-catenin pathway in response to expression of short hairpin RNA (shRNA) against Hsp90α or a control non-targeting shRNA, under the control of a tetracycline-responsive promoter. Hsp90α knockdown contributed to a deregulated stress response, presenting with reduced Hsp27 and β-catenin protein, but corresponded to an increase in the association between Hsp90, β-catenin and Hsp27 in vitro. The reduction of Hsp90α did not influence sensitivity of the colon cancer cells to activators or inhibitors of the Wnt pathway, but rather correlated to reduced TS formation, cell adhesion and spreading, identifying potential therapeutic benefit to the controlled reduction of Hsp90α for the deregulation of colon cancer characteristics. Given that Hsp27 and β-catenin are both involved in cell adhesion, cytoskeletal dynamics and interact directly with each other, we propose a role for targeting Hsp90α in the regulation cell adherence indirectly via reductions in levels of β-catenin and Hsp27, rather than by modifying the transcriptional activity of β-catenin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015-04-09
Systematics and phylogeography of mormyrid fishes in South Africa
- Authors: Maake, Pholoshi Abram
- Date: 2015-04-09
- Subjects: Marcusenius , Fishes Morphology , Petrocephalus , Fishes Phylogeny , Phylogeography , Fishes Identification
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/480323 , vital:78431
- Description: Two genera of mormyrids have been reognised from South Africa. Marcuseniusis the largest genus of the Mormyridae, comprising 38 species distributed throughout tropical and sub-tropical Africa. In southern and eastern Africa eight species have been recognized, including M. pongolensis from South Africa. Petrocephalus has about 30 species distributed mainly in tropical Africa, and 15 of these were recently identified in southern and eastern Africa. This thesis integrated several techniques, through the collection of complementary, but separate data sets to investigate the phylogeography of M. pongolensis in South Africa, and the systematics, evolutionary history, biogeography of Marcusenius and Petrocephalus fishes in southern and eastern Africa. Mitochondrial and nuclear genetic markers consistently demonstrated strong population structuring and identified three divergent lineages within South Africa. The widespread Pongola Lineage occurs partly in the Limpopo River system, and is very common in the Incomati, Pongola (type locality) and Kosi river systems. The Limpopo Lineage endemic only in the Limpopo River system is monophyletic with the Mhlatuze-Nseleni Lineage which is only known from the Mhlatuze and Nseleni rivers in the south. There was strong indication of historical isolation, in the form of allopatric fragmentation between genetically divergent Limpopo and Mhlatuze-Nseleni lineages. High genetic diversity was observed in the Limpopo River system within the Limpopo Lineage and the Pongola Lineage. The occurrence of the Mhlatuze-Nseleni Lineage in the far south cannot be explained by the hypothesis of confluence of rivers, river capture or episodic drainage connections, as the Mhlatuze and Nseleni Rivers would not have had a common confluence or shared low relief terrain with the Limpopo, Incomati, Pongola or Kosi river systems. Morphological studies of these lineages revealed even more differentiations and were described as: Marcusenius krameri sp. nov. for the Limpopo Lineage, and M. caudisquamatus sp. nov. from the Mhlatuze and Nseleni river systems. The specimens from the Ruvuma River were also well differentiated from all southern and eastern African species of Marcusenius, and were therefore recognised as M. lucombesisp. nov. New diagnosis characters of M. pongolensis were re-described and a key to the southern and eastern African species of Marcusenius was also provided. The results of all phylogenetic approaches that included all known southern and eastern African Marcusenius species are highly congruent and provided very strong support for recognizing Marcusenius species from these regions as a monophyletic assemblage that evolved from a single common ancestor. The southern and eastern African Petrocephalus is divided into two deeply mitochondrial and nuclear divergent Western and Eastern groups that are separated by the Victoria Falls. The phylogenetic relationships within each group were not supported and future studies incorporating more molecular characters and species will likely help to resolve the relationships. Speciation within Marcusenius and Petrocephalus ranges in the focus of at least 0.8-5 Million years ago in agreement with geological data, which suggested that vicariant events associated with the formation of the major river systems played a major role in the evolution and distribution of both genera in southern and eastern Africa. The high degree of genetic differentiation and at least four previously undescribed lineages recovered in this study suggested that mormyrid diversity within southern and eastern Africa may be underestimated. Possible threats to the studied mormyrid species and lineages and other freshwater-dependent species were identified during the course of this study. When described, the unidentified lineages would further increase the species diversity of both Marcusenius and Petrocephalus, and, therefore, the southern and eastern African ichthyofauna. , Thesis (PhD) -- Faculty of Science, Ichthyology and Fisheries Science, 2025
- Full Text:
- Date Issued: 2015-04-09
- Authors: Maake, Pholoshi Abram
- Date: 2015-04-09
- Subjects: Marcusenius , Fishes Morphology , Petrocephalus , Fishes Phylogeny , Phylogeography , Fishes Identification
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/480323 , vital:78431
- Description: Two genera of mormyrids have been reognised from South Africa. Marcuseniusis the largest genus of the Mormyridae, comprising 38 species distributed throughout tropical and sub-tropical Africa. In southern and eastern Africa eight species have been recognized, including M. pongolensis from South Africa. Petrocephalus has about 30 species distributed mainly in tropical Africa, and 15 of these were recently identified in southern and eastern Africa. This thesis integrated several techniques, through the collection of complementary, but separate data sets to investigate the phylogeography of M. pongolensis in South Africa, and the systematics, evolutionary history, biogeography of Marcusenius and Petrocephalus fishes in southern and eastern Africa. Mitochondrial and nuclear genetic markers consistently demonstrated strong population structuring and identified three divergent lineages within South Africa. The widespread Pongola Lineage occurs partly in the Limpopo River system, and is very common in the Incomati, Pongola (type locality) and Kosi river systems. The Limpopo Lineage endemic only in the Limpopo River system is monophyletic with the Mhlatuze-Nseleni Lineage which is only known from the Mhlatuze and Nseleni rivers in the south. There was strong indication of historical isolation, in the form of allopatric fragmentation between genetically divergent Limpopo and Mhlatuze-Nseleni lineages. High genetic diversity was observed in the Limpopo River system within the Limpopo Lineage and the Pongola Lineage. The occurrence of the Mhlatuze-Nseleni Lineage in the far south cannot be explained by the hypothesis of confluence of rivers, river capture or episodic drainage connections, as the Mhlatuze and Nseleni Rivers would not have had a common confluence or shared low relief terrain with the Limpopo, Incomati, Pongola or Kosi river systems. Morphological studies of these lineages revealed even more differentiations and were described as: Marcusenius krameri sp. nov. for the Limpopo Lineage, and M. caudisquamatus sp. nov. from the Mhlatuze and Nseleni river systems. The specimens from the Ruvuma River were also well differentiated from all southern and eastern African species of Marcusenius, and were therefore recognised as M. lucombesisp. nov. New diagnosis characters of M. pongolensis were re-described and a key to the southern and eastern African species of Marcusenius was also provided. The results of all phylogenetic approaches that included all known southern and eastern African Marcusenius species are highly congruent and provided very strong support for recognizing Marcusenius species from these regions as a monophyletic assemblage that evolved from a single common ancestor. The southern and eastern African Petrocephalus is divided into two deeply mitochondrial and nuclear divergent Western and Eastern groups that are separated by the Victoria Falls. The phylogenetic relationships within each group were not supported and future studies incorporating more molecular characters and species will likely help to resolve the relationships. Speciation within Marcusenius and Petrocephalus ranges in the focus of at least 0.8-5 Million years ago in agreement with geological data, which suggested that vicariant events associated with the formation of the major river systems played a major role in the evolution and distribution of both genera in southern and eastern Africa. The high degree of genetic differentiation and at least four previously undescribed lineages recovered in this study suggested that mormyrid diversity within southern and eastern Africa may be underestimated. Possible threats to the studied mormyrid species and lineages and other freshwater-dependent species were identified during the course of this study. When described, the unidentified lineages would further increase the species diversity of both Marcusenius and Petrocephalus, and, therefore, the southern and eastern African ichthyofauna. , Thesis (PhD) -- Faculty of Science, Ichthyology and Fisheries Science, 2025
- Full Text:
- Date Issued: 2015-04-09
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