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Title: Evaluation of transcriptional cyclin dependent kinase inhibitors as potential cancer therapeutics
Author: Liu, Xiangrui
ISNI:       0000 0004 2743 1310
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2012
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Cancer cells depend heavily on sustained expression of anti-apoptotic proteins. Targeting transcription to suppress these anti-apoptotic proteins seems a promising strategy for anti-cancer therapy. Cyclin-dependant kinase 9 (CDK9) regulates transcription elongation by phosphorylating Ser2 on the C-terminal domain of RNA polymerase II, while CDK7 phosphorylates Ser5 during transcription initiation. A screening cascade comprised of an MTT assay, a caspase-3 activation assay, a p53 stabilization assay and a mitotic index assay was developed to classify compounds and identify lead transcriptional CDK inhibitors from a novel class of 2,4,5-trisubstituted pyrimidines. Compounds S3-41 and CDKI-71 are the most potent CDK9 inhibitors identified by the screening cascade. They showed potent anti-proliferative activity in the MTT assay and induce both caspase-3 activity and p53 protein level at the GI50 concentration. In addition, no significant effect on mitotic index was observed. The detailed mechanism of action of CDKI-71 was further investigated and compared with the clinical compound, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis that were closely associated with the inhibition of RNAPII phosphorylation at Ser2. This indicated effective targeting of cyclinT-CDK9 and the downstream inhibition of anti-apoptotic proteins such as Mcl-l in cells. Similar to flavopiridol, CDKI-71 down-regulated a large range of genes, including Mcl-l and Bcl-2. No correlation between apoptosis and inhibition of cell cycle CDKs 1 and 2 was observed. Non-transformed lung fibroblast cell lines showed resistance to CDKI-71 treatment. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Flavopiridol also induced genotoxic stress through the induction of DNA double-strand breakage. These results suggest that CDKI-71 has a great potential to be developed as an anti-cancer agent. Another study focused on in vitro anti-tumour mechanism of CDKI-83, a dual inhibitor of CDK9 and CDKI, was performed in A2780 ovarian cancer cells. CDKI-71 presented potent anti-proliferation activity and induced apoptosis in A2780 cells. By inhibiting cellular CDK1 and CDK9 activities, CDKI-83 arrested cells in G2 phase and reduced anti-apoptotic proteins at both mRNA and protein levels, respectively. This study suggests that the combination of CDK9 and CDK1 inhibition results in effective induction of apoptosis in cancer cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP501 Animal biochemistry ; RC 254 Neoplasms. Tumors. Oncology (including Cancer)