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UBC Theses and Dissertations
Role of guanosine triphosphatase regulators in fibroblast transformation and lymphocyte development Klinger, Mark
Abstract
Guanosine triphosphatases (GTPases) are signaling mediators involved in regulation of diverse cellular processes including regulation of the actin cytoskeleton, gene transcription, cell cycle regulation, apoptosis and transformation. Regulatory, proteins including G protein coupled receptors (GPCR), GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF) influence the activity of GTPases. Balanced regulation of GTPase activity is critical in coordinating normal cellular responses. This thesis addresses the contributions of GTPase regulators in cellular growth control, differentiation and transformation. Over-expression of G2A or PAR-1, two GPCRs, in NIH 3T3 fibroblasts induced a full range of phenotypes characteristic of oncogenic transformation. Co-expression of dominant negative Rho or LscRGS (Lbc's second cousin regulator of G protein signaling domain), a negative regulator of Gα₁₂ and Gα₁₃ GTPases, suppressed transformation via these GPCRs. Activation of Gα₁₂, Gα₁₃ and Rho GTPases are thus required for transformation via these GPCRs. Gα₁₂ and Gα₁₃ are unique in that they are activated upstream of Rho. Moreover, Rho GTPase activity is regulated via GEFs. Gct-mediated activation of GEFs and downstream smaller molecular weight GTPases appears to be an important mechanism of linking divergent GTPases downstream of GPCR activation. To elucidate the role of Gα₁₂ and Gα₁₃ GTPases in lymphocyte development, transgenic mice expressing LscRGS were generated. Analyses of lymphocytes from these mice revealed that LscRGS expression did not overtly affect lymphocyte development. These results indicate that Gα₁₂ and Gα₁₃ are not required for lymphocyte development. Rho and Cdc42 are two GTPases involved in lymphocyte development. Previous studies by others demonstrated that loss of Rho function partially blocked differentiation and survival of CD4/CD8- double negative (DN) thymocytes and expression of another Rho family GTPase, Cdc42, enhanced the proliferative capacity of DN thymocytes. In addition, results from other studies revealed that expression of activated Rho augments positive selection and induces CD4⁺/CD8- and CD4/CD8⁺ single positive (SP) thymocyte hypersensitivity to TCR-induced proliferation in vitro. Dbs is a Rho- and Cdc42-activating GEF normally expressed in thymus. To determine how Dbs influences lymphocyte development, transgenic mice were generated expressing an activated form of Dbs. Expression of activated Dbs in lymphocytes promoted the accumulation of early thymocytes and restricted the production of mature thymocytes. Activated Dbs expression also led to increased proliferation of DN thymocytes. The Dbs transgene caused reduced numbers of SP thymocytes and mature splenic T lymphocytes. In addition, transgenic CD4⁺/CD8⁺ double positive (DP) thymocytes expressed higher levels of T cell receptor (TCR) and were hypersensitive to apoptosis induced by injection of anti-CD3. Moreover, Dbs transgenic thymocytes displayed impaired positive selection. Thymocyte culture experiments revealed that proliferation in response to anti-CD3 was reduced in SP thymocytes from Dbs transgenic mice. Expression of activated Dbs, a positive regulator of Rho and Cdc42, promoted the accumulation of DN thymocytes; this is the opposite of the DN phenotype observed in thymocytes lacking Rho function and similar to the phenotype displayed by Cdc42 transgenic mice. Thus the accumulation of DN thymocytes is likely to occur via Rho and/or Cdc42 activation. Activated Dbs expression also caused reduced in vitro SP thymocyte proliferation in response to TCR cross-linking and impaired thymocyte positive selection. These results are contrary to previous reports describing transgenic mice expressing activated Rho; thus, impaired thymocyte proliferation and positive selection in Dbs transgenic mice is likely to involve a pathway independent of Rho. Results presented in this thesis provide insights into the contributions of GTPase regulators in regulation of cellular growth control, differentiation and transformation.
Item Metadata
Title |
Role of guanosine triphosphatase regulators in fibroblast transformation and lymphocyte development
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2002
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Description |
Guanosine triphosphatases (GTPases) are signaling mediators involved in regulation of diverse cellular
processes including regulation of the actin cytoskeleton, gene transcription, cell cycle regulation,
apoptosis and transformation. Regulatory, proteins including G protein coupled receptors (GPCR),
GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF) influence the activity
of GTPases. Balanced regulation of GTPase activity is critical in coordinating normal cellular responses.
This thesis addresses the contributions of GTPase regulators in cellular growth control, differentiation and
transformation.
Over-expression of G2A or PAR-1, two GPCRs, in NIH 3T3 fibroblasts induced a full range of phenotypes
characteristic of oncogenic transformation. Co-expression of dominant negative Rho or LscRGS (Lbc's
second cousin regulator of G protein signaling domain), a negative regulator of Gα₁₂ and Gα₁₃ GTPases,
suppressed transformation via these GPCRs. Activation of Gα₁₂, Gα₁₃ and Rho GTPases are thus
required for transformation via these GPCRs. Gα₁₂ and Gα₁₃ are unique in that they are activated
upstream of Rho. Moreover, Rho GTPase activity is regulated via GEFs. Gct-mediated activation of
GEFs and downstream smaller molecular weight GTPases appears to be an important mechanism of
linking divergent GTPases downstream of GPCR activation.
To elucidate the role of Gα₁₂ and Gα₁₃ GTPases in lymphocyte development, transgenic mice expressing
LscRGS were generated. Analyses of lymphocytes from these mice revealed that LscRGS expression
did not overtly affect lymphocyte development. These results indicate that Gα₁₂ and Gα₁₃ are not
required for lymphocyte development.
Rho and Cdc42 are two GTPases involved in lymphocyte development. Previous studies by others
demonstrated that loss of Rho function partially blocked differentiation and survival of CD4/CD8- double
negative (DN) thymocytes and expression of another Rho family GTPase, Cdc42, enhanced the
proliferative capacity of DN thymocytes. In addition, results from other studies revealed that expression of
activated Rho augments positive selection and induces CD4⁺/CD8- and CD4/CD8⁺ single positive (SP)
thymocyte hypersensitivity to TCR-induced proliferation in vitro. Dbs is a Rho- and Cdc42-activating GEF
normally expressed in thymus. To determine how Dbs influences lymphocyte development, transgenic
mice were generated expressing an activated form of Dbs. Expression of activated Dbs in lymphocytes
promoted the accumulation of early thymocytes and restricted the production of mature thymocytes.
Activated Dbs expression also led to increased proliferation of DN thymocytes. The Dbs transgene
caused reduced numbers of SP thymocytes and mature splenic T lymphocytes. In addition, transgenic
CD4⁺/CD8⁺ double positive (DP) thymocytes expressed higher levels of T cell receptor (TCR) and were
hypersensitive to apoptosis induced by injection of anti-CD3. Moreover, Dbs transgenic thymocytes
displayed impaired positive selection. Thymocyte culture experiments revealed that proliferation in
response to anti-CD3 was reduced in SP thymocytes from Dbs transgenic mice. Expression of activated
Dbs, a positive regulator of Rho and Cdc42, promoted the accumulation of DN thymocytes; this is the
opposite of the DN phenotype observed in thymocytes lacking Rho function and similar to the phenotype
displayed by Cdc42 transgenic mice. Thus the accumulation of DN thymocytes is likely to occur via Rho
and/or Cdc42 activation. Activated Dbs expression also caused reduced in vitro SP thymocyte
proliferation in response to TCR cross-linking and impaired thymocyte positive selection. These results
are contrary to previous reports describing transgenic mice expressing activated Rho; thus, impaired
thymocyte proliferation and positive selection in Dbs transgenic mice is likely to involve a pathway
independent of Rho. Results presented in this thesis provide insights into the contributions of GTPase
regulators in regulation of cellular growth control, differentiation and transformation.
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Extent |
26793216 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-10-02
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0090682
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2002-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.