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postgraduate thesis: Functional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery system

TitleFunctional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery system
Authors
Issue Date2011
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Xu, Z. [许振华]. (2011). Functional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery system. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775265
AbstractCell cycle-related kinase (CCRK) is a 42 KDa serine/threonine protein kinase homologous to Cdk1, 2 and 7. Previous work has shown that CCRK regulates cell cycle transition by phosphorylating Cdk2 and Rb. More importantly, it was found that CCRK was a candidate oncogene in both glioblastoma multiform (GBM) and human colorectal cancer. However, the mechanistic role of CCRK in tumorigenicity is still not completely understood. In the first part of this thesis, I found that casein kinas II beta (CKIIβ) was one of proteins that interact with CCRK using the high-throughput yeast-two-hybrid analysis. Then I confirmed their interaction by co-immunoprecipitation. CCRK phosphorylated CKIIβ at Ser-209 in a cell cycle-dependent manner. The phosphorylation of CKIIβ by CCRK enhanced the activity of CKII holoenzyme, protected CKIIβ against proteasome degradation, and facilitated CKIIβ translocation into the nucleus in U-87 MG and U-373 MG GBM cells. Importantly, CCRK de-sensitized GBM cells to the cytotoxic effect of three chemotherapy drugs, whereas knockdown of CCRK by siRNA reduced chemoresistance. Functionally, CKIIβ is responsible for CCRK-mediated inhibition of apoptosis, as suppression of CKIIβ by siRNA or CKIIβ inhibitor could re-sensitize cells to the cytotoxic effect of cisplatin in both wild type and CCRK-overexpressing U-87 MG cells. In vivo studies also showed that stable over-expression of CCRK increased tumor growth and decreased the anti-tumor efficacy of cisplatin in a nude mice GBM xenograft model. These results provide the first evidence that phosphorylation of CKIIβ is a new mechanism by which CCRK confers tumor growth and drug resistance to GBM cells. In the second part of this thesis I described a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600 and sebacoyl chloride. 1H-NMR, FT-IR and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. Cytotoxicity of the mPPS-FA/DNA nanoparticles was also evaluated on B16-F0, U87MG, CHO-1 and Ho-8910 cells. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910 and A549 cells was investigated in vitro as compared to the lipid-based transfection agent LipofectamineTM2000 and Linear PEI 22KDa (L-PEI 22KDa). I found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1 and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be significantly competed and blocked by the free folic acid molecules. All together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier.
DegreeDoctor of Philosophy
SubjectProtein kinases.
Cell cycle.
Gliomas.
Genetic vectors.
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/181852

 

DC FieldValueLanguage
dc.contributor.authorXu, Zhenhua-
dc.contributor.author许振华-
dc.date.accessioned2013-03-20T06:29:00Z-
dc.date.available2013-03-20T06:29:00Z-
dc.date.issued2011-
dc.identifier.citationXu, Z. [许振华]. (2011). Functional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery system. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775265-
dc.identifier.urihttp://hdl.handle.net/10722/181852-
dc.description.abstractCell cycle-related kinase (CCRK) is a 42 KDa serine/threonine protein kinase homologous to Cdk1, 2 and 7. Previous work has shown that CCRK regulates cell cycle transition by phosphorylating Cdk2 and Rb. More importantly, it was found that CCRK was a candidate oncogene in both glioblastoma multiform (GBM) and human colorectal cancer. However, the mechanistic role of CCRK in tumorigenicity is still not completely understood. In the first part of this thesis, I found that casein kinas II beta (CKIIβ) was one of proteins that interact with CCRK using the high-throughput yeast-two-hybrid analysis. Then I confirmed their interaction by co-immunoprecipitation. CCRK phosphorylated CKIIβ at Ser-209 in a cell cycle-dependent manner. The phosphorylation of CKIIβ by CCRK enhanced the activity of CKII holoenzyme, protected CKIIβ against proteasome degradation, and facilitated CKIIβ translocation into the nucleus in U-87 MG and U-373 MG GBM cells. Importantly, CCRK de-sensitized GBM cells to the cytotoxic effect of three chemotherapy drugs, whereas knockdown of CCRK by siRNA reduced chemoresistance. Functionally, CKIIβ is responsible for CCRK-mediated inhibition of apoptosis, as suppression of CKIIβ by siRNA or CKIIβ inhibitor could re-sensitize cells to the cytotoxic effect of cisplatin in both wild type and CCRK-overexpressing U-87 MG cells. In vivo studies also showed that stable over-expression of CCRK increased tumor growth and decreased the anti-tumor efficacy of cisplatin in a nude mice GBM xenograft model. These results provide the first evidence that phosphorylation of CKIIβ is a new mechanism by which CCRK confers tumor growth and drug resistance to GBM cells. In the second part of this thesis I described a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600 and sebacoyl chloride. 1H-NMR, FT-IR and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. Cytotoxicity of the mPPS-FA/DNA nanoparticles was also evaluated on B16-F0, U87MG, CHO-1 and Ho-8910 cells. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910 and A549 cells was investigated in vitro as compared to the lipid-based transfection agent LipofectamineTM2000 and Linear PEI 22KDa (L-PEI 22KDa). I found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1 and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be significantly competed and blocked by the free folic acid molecules. All together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47752658-
dc.subject.lcshProtein kinases.-
dc.subject.lcshCell cycle.-
dc.subject.lcshGliomas.-
dc.subject.lcshGenetic vectors.-
dc.titleFunctional characterization of cell cycle-related kinase in glioblastoma and development of gene delivery system-
dc.typePG_Thesis-
dc.identifier.hkulb4775265-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4775265-
dc.date.hkucongregation2012-

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