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Development and Application of a Cellular RR Assay for Clinical Cancer Research

Author: JiangHongJuan
Tutor: ShaoJiMin
School: Zhejiang University
Course: Pathology and Pathophysiology
Keywords: Ribonucleotide reductase Enzymatic assay development Application
CLC: R73-3
Type: Master's thesis
Year: 2011
Downloads: 21
Quote: 0
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Abstract


Ribonucleotide reductase (RR) is a multisubunit enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides, which are building blocks for DNA replication and repair. Inhibition of RR will lead to a decrease in DNA synthesis and ultimately cell death. Human RR consists of two dimers:RRM1 (large subunit) and RRM2 or RRM2B (small subunit), which constitut two forms of RR in human cells (RRM1/RRM2 and RRM1/RRM2B). RRM1 contains the active site and binding sites for allosteric effectors. RRM2 and RRM2B are homologues, both possess a diiron-tyrosyl radical co-factor that is essential for enzyme activity. Increased RR activity is directly involved in tumor growth, metastasis, and drug resistance. Therefore, small-molecule inhibitors of RR have been widely investigated as potential anti-tumoral chemotherapeutic agents.Hydroxyurea blocks RR activity and hence DNA synthesis by reducing the tyrosyl radical and perturbing the iron center of the small subunits. Once the RR activity is inhibited by HU, DNA synthesis will be blocked and the cells will subsequently be killed. However, HU therapy rapidly induces the resistance of cancer cells to the drug. Triapine belongs to a class of powerful iron chelators. Triapine-Fe(Ⅱ) is much more effective at inhibiting RRM1/RRM2B by generating ROS in cells. Triapine possesses 100～1000-fold higher potency in both enzyme and tumor cell growth inhibition than hydroxyurea.The compound is fully active against hydroxyurea-resistant tumors. DFO (Desferrioxamine) is another kind of iron chelators, it inhibits RR through chelating intracellular iron pools and preventing the activation and regeneration of the enzymatic activity.In previous study, we established an in vitro recombinant RR activity assay using RR subunit proteins (RRM1, RRM2 and RRM2B) expressed in E.coli for the study of the structure and function of RR and screening of new anti-cancer drugs in vitro. In this study, we have developed a standard cellular RR activity assay using crude protein extracts from cultured cells and clinical tissures combined with qRT-PCR and western blot methords to analyze the expression and activity of RR clinical tumor tissures, evaluate the efficiency of RR inhibitor, and clarify the mechanism of RR inhibitor resistanc.(1) Cellular prtein extracts, RR assay development and standerization:cellar proteins were extracted form cellar homogenates after treated with 1% streptomycin and 40% saturated ammonium sulfate. Then, the cellular RR assay was development based CDP reduction activity and standerized(2) Assessment of RR activity and expression level in clinical cancer tissures: crude protein extracts were prepared from normal and cancer tissues of liver and colon for assessment wity, the established cellular RR assay and western blot. It was revealed that compared to the normal tissures, RR activity in cancer tissues were overall increased with variations in different cancers as well as in different cases, and the protein levels of RRM1, RRM2 and RRM2B were also changed differently in normal and cancer tissues. The primary data demonstrates the usefulness of the established cellular RR assay combined with other methords for systemic study of the mechanisms of RR involved in cancer development.(3) RR inhibitor evaluation with the established cellular RR assay:the efficiency of DFO、BSO、HU and 3-AP to inhibit RR activity in protein extracts from KB、MCF-7 and colon cancer tissues were evaluated with the established cellular RR assay. DFO and BSO were showed to have inhibitory potency with IC50s about 50 mM. These data indicate that the established cellular RR assay may be used to assess RR inhibitor activity in clinical cancer tissurs, which would be helpful for cancer personalized medicine.(4) The mechanism of RR inhibitor resistanc analysis:Hu-resistant human nasopharyngeal carcinoma KB cells (KB-HU) were developed with stepwised addition of Hu in KB cell cultures. Real-time RT-PCR measurements showed that KB-HU cells exhibited a 3-fold elevation of RRM2 mRNA over KB cells, but no difference of RRM1 and RRM2B mRNA between KB and KB-HU cells. Western blot analysis revealed that RRM2 proteins were increased in KB-HU compared to KB cells, while the protein level of RRM1 and RRM2B had no significant difference between KB and KB-Hu cells. RR activity assays showed that RR activity of KB-HU cell was significantly increased compared with KB cell. These data showed that the alteration in mRNA and protein levels of RRM2 but not RRM2B or M1 was involved in the increased RR activity and HU resistance phenotype, which may partially explain the molecular mechanism of HU resistance in the KB-HU cells.Conclusion:In this study, we have established and standerized a cellular RR assay combined with other quantitative methords for clinical research on the mechanisms of RR involved in cancer development and evaluation of RR inhibitor efficiency in vitro for caner personalized medicine.

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