Cell 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. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/181852 |
| Date | January 2011 |
| Creators | Xu, Zhenhua, 许振华 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47752658 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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