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

Abstract

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.