Expression of heat shock proteins on the plasma membrane of cancer cells : a potential multi-chaperone complex that mediates migration
- Authors: Kenyon, Amy
- Date: 2011 , 2011-03-29
- Subjects: Heat shock proteins , Protein folding , Molecular chaperones , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4122 , http://hdl.handle.net/10962/d1013362
- Description: Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.
- Full Text:
- Date Issued: 2011
- Authors: Kenyon, Amy
- Date: 2011 , 2011-03-29
- Subjects: Heat shock proteins , Protein folding , Molecular chaperones , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4122 , http://hdl.handle.net/10962/d1013362
- Description: Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.
- Full Text:
- Date Issued: 2011
The relationship between three anxiety related clusters in projective drawings and anxiety and ego-strength scales of the Minnesota multiphasic personality inventory-2
- Authors: Brink, André
- Date: 2002
- Subjects: Projective techniques , Personality assessment , Personality tests , Anxiety , Minnesota Multiphasic Personality Inventory
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:2942 , http://hdl.handle.net/10962/d1002451 , Projective techniques , Personality assessment , Personality tests , Anxiety , Minnesota Multiphasic Personality Inventory
- Description: Based on the shortcomings of past research, the need for understanding and investigation of the general relationship between self-report measures and human figure drawings required understanding and investigation (Riethmiller & Handler, 1997b; Waehler, 1997) while utilising a quantitative, configural scoring approach. Riethmiller and Handler (1997a; 1997b) hypothesised that subjects have one of two typical approach styles to anxiety/stress that influences their execution of the Human Figure Drawing (HFD) Test: “Avoidance” or “Coping” as measured by composite scoring index clusters. They argue that these two approach styles had to be taken into account when investigating anxiety on the HFD Test. According to Handler and Reyher (1965) those who experience more intense anxiety typically rely on an “Avoidant” approach, while those with lower anxiety typically rely on a “Coping” approach. The “Coping” response is hypothesised to suggest good ego-strength, and the “Avoidant” response poor ego-strength. Handler and Reyher (1964; 1965; 1966) also argued that there are two sources of anxiety on projective drawings: internal and external sources of anxiety. They hypothesised that the “External” anxiety cluster (measured by utilising the car drawing) and self-report measures both assess ‘external’ anxiety. Using Handler’s (1967) HFD index scoring manual, this research therefore inve stigated the level of correlation of the two MMPI-2 anxiety scale scores with (a) the hypothesised Stress Approach HFD cluster scores, as well as with (b) the “External” anxiety cluster score, while the hypothesised Stress Approach HFD cluster scores were compared with the (c) MMPI-2 ego strength scale score. The results of the investigated relationships yielded non-significant correlations overall. The differences in nature of the two measurement instruments, and the potential weaknesses of this study, as two likely explanations for these correlations, are discussed. In the consideration of the differences of the two measurement instruments, the weaknesses of SR measures and criterion-related validity are discussed while self- attributed and implicit motives are contrasted with each other. Potential extraneous variables and possible truncated range are discussed as potential weaknesses of this study.
- Full Text:
- Date Issued: 2002
- Authors: Brink, André
- Date: 2002
- Subjects: Projective techniques , Personality assessment , Personality tests , Anxiety , Minnesota Multiphasic Personality Inventory
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:2942 , http://hdl.handle.net/10962/d1002451 , Projective techniques , Personality assessment , Personality tests , Anxiety , Minnesota Multiphasic Personality Inventory
- Description: Based on the shortcomings of past research, the need for understanding and investigation of the general relationship between self-report measures and human figure drawings required understanding and investigation (Riethmiller & Handler, 1997b; Waehler, 1997) while utilising a quantitative, configural scoring approach. Riethmiller and Handler (1997a; 1997b) hypothesised that subjects have one of two typical approach styles to anxiety/stress that influences their execution of the Human Figure Drawing (HFD) Test: “Avoidance” or “Coping” as measured by composite scoring index clusters. They argue that these two approach styles had to be taken into account when investigating anxiety on the HFD Test. According to Handler and Reyher (1965) those who experience more intense anxiety typically rely on an “Avoidant” approach, while those with lower anxiety typically rely on a “Coping” approach. The “Coping” response is hypothesised to suggest good ego-strength, and the “Avoidant” response poor ego-strength. Handler and Reyher (1964; 1965; 1966) also argued that there are two sources of anxiety on projective drawings: internal and external sources of anxiety. They hypothesised that the “External” anxiety cluster (measured by utilising the car drawing) and self-report measures both assess ‘external’ anxiety. Using Handler’s (1967) HFD index scoring manual, this research therefore inve stigated the level of correlation of the two MMPI-2 anxiety scale scores with (a) the hypothesised Stress Approach HFD cluster scores, as well as with (b) the “External” anxiety cluster score, while the hypothesised Stress Approach HFD cluster scores were compared with the (c) MMPI-2 ego strength scale score. The results of the investigated relationships yielded non-significant correlations overall. The differences in nature of the two measurement instruments, and the potential weaknesses of this study, as two likely explanations for these correlations, are discussed. In the consideration of the differences of the two measurement instruments, the weaknesses of SR measures and criterion-related validity are discussed while self- attributed and implicit motives are contrasted with each other. Potential extraneous variables and possible truncated range are discussed as potential weaknesses of this study.
- Full Text:
- Date Issued: 2002
Proportional distribution of predominant rumen bacteria between the solid and the liquid portions of ruminal ingesta
- Authors: Brinkman, Paul A
- Date: 1966
- Subjects: Rumen -- Microbiology , Bacteria -- Rumen
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4100 , http://hdl.handle.net/10962/d1009715 , Rumen -- Microbiology , Bacteria -- Rumen
- Description: That certain bacteria in the rumen of sheep and cattle are attached to solid particles in the ruminal ingesta has been known for many years. In 1942 Baker published direct microscopical evidence that bacteria were attached to cellulose food particles and to starch granules in the rumen. The sites of attachment of these bacteria corresponded to sites of disintegration of the particles when viewed by polarised light. This indicated that at least bacteria attacking solid substrates such as cellulose and starch were attached to particles of ruminal ingesta. Van der Wath (1942) found rumen bacteria attached to particles of chemically pure cellulose and of crushed maize which he suspended in separate compartments of a pure silk bag inside the rumen of sheep. The bacteria associated with the particles of cellulose were mainly Gram negative rods , while clusters of iodophilic cocci were observed in most instances around the maize kernels . The latter organisms were isolated in pure culture and found to be heat-tolerant, short-chain, Gram positive cocci fermenting glucose, maltose, and other soluble sugars as well as starch. It was thus not surprising that many years later Schwartz et al (1964) obtained evidence which suggested that bacteria metabolising soluble substrates such as glucose also showed marked attachment to solid particles of ingesta.
- Full Text:
- Date Issued: 1966
- Authors: Brinkman, Paul A
- Date: 1966
- Subjects: Rumen -- Microbiology , Bacteria -- Rumen
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4100 , http://hdl.handle.net/10962/d1009715 , Rumen -- Microbiology , Bacteria -- Rumen
- Description: That certain bacteria in the rumen of sheep and cattle are attached to solid particles in the ruminal ingesta has been known for many years. In 1942 Baker published direct microscopical evidence that bacteria were attached to cellulose food particles and to starch granules in the rumen. The sites of attachment of these bacteria corresponded to sites of disintegration of the particles when viewed by polarised light. This indicated that at least bacteria attacking solid substrates such as cellulose and starch were attached to particles of ruminal ingesta. Van der Wath (1942) found rumen bacteria attached to particles of chemically pure cellulose and of crushed maize which he suspended in separate compartments of a pure silk bag inside the rumen of sheep. The bacteria associated with the particles of cellulose were mainly Gram negative rods , while clusters of iodophilic cocci were observed in most instances around the maize kernels . The latter organisms were isolated in pure culture and found to be heat-tolerant, short-chain, Gram positive cocci fermenting glucose, maltose, and other soluble sugars as well as starch. It was thus not surprising that many years later Schwartz et al (1964) obtained evidence which suggested that bacteria metabolising soluble substrates such as glucose also showed marked attachment to solid particles of ingesta.
- Full Text:
- Date Issued: 1966
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