| dc.identifier.uri | http://hdl.handle.net/11401/76895 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Thesis | |
| dcterms.abstract | Abstract of the Thesis Effects of Lentiviral STAT3 and STAT3 Mutants on Cellular Proliferation and Tumor Formation by Alkiviadis Stavros Pierrakeas Master of Science in Biochemistry and Cell Biology Stony Brook University 2016 The mammalian Signal Transducers and Activators of Transcription (STAT) family of proteins includes seven STAT transcription factors that function in cytokine signaling pathways to activate gene expression affecting cell proliferation, cell growth, cell survival, and apoptosis. Cytokines such as interleukin-6 (IL-6), and several growth factors are able to bind to cell surface receptors, activate tyrosine kinase activity and promote downstream phosphorylation of STAT proteins. Cytokines associated with Janus kinases (JAKs) classically activate what has been termed JAK/STAT signaling pathways. Tyrosine-phosphorylated STATs are able to dimerize, enter the nucleus, and function by binding DNA and regulating gene transcription. One of the most well studied STAT proteins is STAT3, a necessary transcription factor for embryonic development and a critical component in various signaling mechanisms. STAT3 structurally and functionally resembles other members of the STAT family. Its activation requires phosphorylation of a specific tyrosine residue (Y705), and its activity can be enhanced even further by an additional serine residue phosphorylation (S727). Previous studies have shown that constitutively active STAT3 is present in many different cancers ranging from solid tumors (in the pancreas, prostate, mammary glands, lungs, etc.) to hematological cancers (including lymphomas and leukemia). Constitutive activation of STAT3 is believed to stimulate cell proliferation, differentiation, and transformation of cells, while also repressing apoptosis, and thus, promote tumorigenesis. Defects in several stages of the JAK/STAT3 signaling pathway, including inactivating mutations in negative feedback regulators and hyperactivating mutations in upstream tyrosine kinases (JAKs), can produce constitutively active STAT3 proteins. STAT3 has also been found to function as a tumor suppressor in some cases, with inactivation of STAT3 resulting in more prominent tumor phenotypes (lung tissue). The objective of my studies was to understand how STAT3 functions in different cells to either promote or inhibit tumorigenesis. The approach was to generate lentiviruses expressing STAT3, express lentiviral STAT3 in pre-malignant cells, and evaluate the proliferation and xenograph tumor formation of the cells. I created lentiviruses expressing wild-type (WT) STAT3 or one of several mutant STAT3 genes. We then ectopically expressed WT STAT3 or mutant STAT3 in pre-malignant pancreatic cells and pre-malignant lung cells. The cells were harvested from a mouse with p53 -/-; Lox-Stop-Lox K-RasG12D, and retroviral Cre was used to activate the K-Ras oncogenic mutation. The cells overexpressing WT STAT3 or mutant STAT3 were evaluated for cell behavior and proliferation, tumor growth, and tumor morphology. Both loss-of-function and gain-of-function STAT3 mutations were used to evaluate possible tumor-promoting or tumor-suppressing effects utilizing in vitro and in vivo analyses. Results from our experiments depended on the tissue type, but in each case, STAT3 overexpression did not affect growth in tissue culture. However, when xenograph tumors were evaluated, STAT3 was found to promote differentiation. Pancreatic cells overexpressing WT STAT3 generated tumors more rapidly, but unexpectedly, histological evaluation showed extensive differentiation with more abundant glandular structures present after hematoxylin & eosin (H&E) and immunohistochemical (IHC) staining. Pancreatic cells expressing mutant STAT3 had similar growth rates as control cells, but they were also more differentiated. The lung cells were notably different. Lentiviral WT STAT3 reduced the rate of tumor growth, as did the STAT3 gain-of-function mutation. Cells with loss-of-function mutant STAT3 had similar growth rates as control. Regardless of the growth rate of lung cell tumors, they were also more differentiated, exhibiting a robust squamous cell carcinoma phenotype after staining tumor sections with H&E and antibodies for IHC. Additional studies are required in order to verify these conclusions, with several other cell lines yet to be examined and other mutant STAT3 constructs still requiring testing. Understanding how activation levels and concentration of STAT3 affect cellular properties and tumor morphology will provide information on how to prevent STAT3’s function in tumorigenesis, perhaps garnering a path for which drugs can be designed to limit STAT3 activity and/or decrease STAT3 concentration in tumor cells. | |
| dcterms.abstract | Abstract of the Thesis Effects of Lentiviral STAT3 and STAT3 Mutants on Cellular Proliferation and Tumor Formation by Alkiviadis Stavros Pierrakeas Master of Science in Biochemistry and Cell Biology Stony Brook University 2016 The mammalian Signal Transducers and Activators of Transcription (STAT) family of proteins includes seven STAT transcription factors that function in cytokine signaling pathways to activate gene expression affecting cell proliferation, cell growth, cell survival, and apoptosis. Cytokines such as interleukin-6 (IL-6), and several growth factors are able to bind to cell surface receptors, activate tyrosine kinase activity and promote downstream phosphorylation of STAT proteins. Cytokines associated with Janus kinases (JAKs) classically activate what has been termed JAK/STAT signaling pathways. Tyrosine-phosphorylated STATs are able to dimerize, enter the nucleus, and function by binding DNA and regulating gene transcription. One of the most well studied STAT proteins is STAT3, a necessary transcription factor for embryonic development and a critical component in various signaling mechanisms. STAT3 structurally and functionally resembles other members of the STAT family. Its activation requires phosphorylation of a specific tyrosine residue (Y705), and its activity can be enhanced even further by an additional serine residue phosphorylation (S727). Previous studies have shown that constitutively active STAT3 is present in many different cancers ranging from solid tumors (in the pancreas, prostate, mammary glands, lungs, etc.) to hematological cancers (including lymphomas and leukemia). Constitutive activation of STAT3 is believed to stimulate cell proliferation, differentiation, and transformation of cells, while also repressing apoptosis, and thus, promote tumorigenesis. Defects in several stages of the JAK/STAT3 signaling pathway, including inactivating mutations in negative feedback regulators and hyperactivating mutations in upstream tyrosine kinases (JAKs), can produce constitutively active STAT3 proteins. STAT3 has also been found to function as a tumor suppressor in some cases, with inactivation of STAT3 resulting in more prominent tumor phenotypes (lung tissue). The objective of my studies was to understand how STAT3 functions in different cells to either promote or inhibit tumorigenesis. The approach was to generate lentiviruses expressing STAT3, express lentiviral STAT3 in pre-malignant cells, and evaluate the proliferation and xenograph tumor formation of the cells. I created lentiviruses expressing wild-type (WT) STAT3 or one of several mutant STAT3 genes. We then ectopically expressed WT STAT3 or mutant STAT3 in pre-malignant pancreatic cells and pre-malignant lung cells. The cells were harvested from a mouse with p53 -/-; Lox-Stop-Lox K-RasG12D, and retroviral Cre was used to activate the K-Ras oncogenic mutation. The cells overexpressing WT STAT3 or mutant STAT3 were evaluated for cell behavior and proliferation, tumor growth, and tumor morphology. Both loss-of-function and gain-of-function STAT3 mutations were used to evaluate possible tumor-promoting or tumor-suppressing effects utilizing in vitro and in vivo analyses. Results from our experiments depended on the tissue type, but in each case, STAT3 overexpression did not affect growth in tissue culture. However, when xenograph tumors were evaluated, STAT3 was found to promote differentiation. Pancreatic cells overexpressing WT STAT3 generated tumors more rapidly, but unexpectedly, histological evaluation showed extensive differentiation with more abundant glandular structures present after hematoxylin & eosin (H&E) and immunohistochemical (IHC) staining. Pancreatic cells expressing mutant STAT3 had similar growth rates as control cells, but they were also more differentiated. The lung cells were notably different. Lentiviral WT STAT3 reduced the rate of tumor growth, as did the STAT3 gain-of-function mutation. Cells with loss-of-function mutant STAT3 had similar growth rates as control. Regardless of the growth rate of lung cell tumors, they were also more differentiated, exhibiting a robust squamous cell carcinoma phenotype after staining tumor sections with H&E and antibodies for IHC. Additional studies are required in order to verify these conclusions, with several other cell lines yet to be examined and other mutant STAT3 constructs still requiring testing. Understanding how activation levels and concentration of STAT3 affect cellular properties and tumor morphology will provide information on how to prevent STAT3’s function in tumorigenesis, perhaps garnering a path for which drugs can be designed to limit STAT3 activity and/or decrease STAT3 concentration in tumor cells. | |
| dcterms.available | 2017-09-20T16:51:23Z | |
| dcterms.contributor | Reich, Nancy C. | en_US |
| dcterms.contributor | Krug, Laurie T.. | en_US |
| dcterms.creator | Pierrakeas, Alkiviadis Stavros | |
| dcterms.dateAccepted | 2017-09-20T16:51:23Z | |
| dcterms.dateSubmitted | 2017-09-20T16:51:23Z | |
| dcterms.description | Department of Biochemistry and Cell Biology | en_US |
| dcterms.extent | 62 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/76895 | |
| dcterms.issued | 2016-12-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:23Z (GMT). No. of bitstreams: 1
Pierrakeas_grad.sunysb_0771M_13161.pdf: 2928665 bytes, checksum: c5e57e7b491e49f613b269cb049d3995 (MD5)
Previous issue date: 1 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Biochemistry -- Molecular biology -- Genetics | |
| dcterms.subject | Adenocarcinoma, Growth, Lentivirus, Morphology, Squamous Cell Carcinoma, STAT3 | |
| dcterms.title | Effects of Lentiviral STAT3 and STAT3 Mutants on Cellular Proliferation and Tumor Formation | |
| dcterms.type | Thesis | |
