Glioblastoma multiforme (GBMs) are the most common and severe form of malignant brain tumors. Despite recent advancement in the fields of surgical resection, radiotherapy and chemotherapy, the prognosis for patients with GBMs remains poor with the median survival rate of approximately a year. Recently, our laboratory has demonstrated the oncogenic role of cell cycle-related kinase (CCRK), a 42-kD protein kinase responsible for regulating cell growth in GBM carcinogenesis, suggesting that CCRK is a candidate oncogene in GBMs. Nevertheless, the regulation of CCRK expression and the cellular mechanism for its overexpression in GBMs remain elusive. Understanding the regulation of human CCRK expression in GBMs should therefore shed light on the development of better prognostic and therapeutic methods for this deadly disease.
This study aims to characterize the human CCRK gene and the regulation of its expression in GBMs. We first characterized the 5’ upstream sequence of CCRK by in silico analysis, which revealed the absence of TATA box but the presence of three potential transcription factor binding sites for Sp1, c-Myc and CREB, and identified the transcription start site by 5’-RACE at 240 bp upstream of the start codon. In vitro analysis of the CCRK promoters revealed the presence of nucleotide polymorphisms in three high-grade glioblastoma cell lines U-87 MG, U-138 MG and U-373 MG, and the control fibroblast cell line CCD19Lu. Furthermore, three CpG islands within the CCRK promoter were identified and the CCRK promoter was hypomethylated in these cell lines. Sp1, c-Myc and CREB binding sites as well as the nucleotide polymorphisms on the CCRK promoter were further investigated. The results from electrophoretic mobility shift assay showed that these transcription factors interacted with the corresponding cis-regulatory elements on the CCRK promoter, removal of the potential Sp1 and c-Myc binding sites lowered the CCRK promoter activity by 46 – 66 % in vitro. In addition, mutations introduced to the nucleotide polymorphisms reduced the CCRK promoter activity by 62 – 81 % in U-87 MG cells and enhanced the CCRK promoter activity by 1.35-fold in U-138 MG cells, suggesting their importance in regulating CCRK expression.
c-Myc is a proto-oncogene with its overexpression associated with a large variety of tumors. As c-Myc binding site was identified in the CCRK promoter, therefore, the effect of c-Myc on CCRK expression was examined. c-Myc overexpression resulted in significant enhancements in the CCRK promoter activity by 30.74-fold in U-87 MG cells, 26.5-fold in U-373 MG cells and 6.09-fold in U-138 MG cells. On the contrary, c-Myc knockdown reduced the CCRK promoter activity by 49 % in U-87 MG cells, 35 % in U-373 MG cells and 17% in U-138 MG cells.
In summary, this study represents the first molecular characterization of the human CCRK gene and findings of this study would prime others for future research on the molecular pathogenesis of CCRK-mediated GBMs and for developing CCRK as a potential therapeutic and diagnostic target for GBMs and possibly other cancers. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/182308 |
| Date | January 2011 |
| Creators | Chu, Ying-ying, Jamie., 朱盈盈. |
| 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/B47849411 |
| 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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