• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2121-2127
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• 2012

Objective: Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reported to exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate the capacity of the mutanted enzyme and its potential for inhibiting cancer cell growth. Methods: We altered the sequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed active site mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expression of lentivirus mediated Dm-dNKmut in a breast cancer cell line (Bcap37), a gastric cancer cell line (SGC7901) and a colorectal cancer cell line (CCL187). [3H]-labeled substrates were used for enzyme activity assays, cell cytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis induction by thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nude mice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidine nucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine). Results: The Dm-dNKmut could be stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressing Dm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitro and in vivo. Conclusion: These findings underlined the importance of BVDU phosphorylated by Dm-dNKmut in transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis for future intensive research for cancer therapy.

• The Korean Journal of Pesticide Science
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• v.5 no.3
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• pp.1-11
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• 2001

New technologies will have a large impact on the discovery of new herbicide site of action. Genomics, combinatorial chemistry, and bioinformatics help take advantage of serendipity through tile sequencing of huge numbers of genes or the synthesis of large numbers of chemical compounds. There are approximately $10^{30}\;to\;10^{50}$ possible molecules in molecular space of which only a fraction have been synthesized. Combining this potential with having access to 50,000 plant genes in the future elevates tile probability of discovering flew herbicidal site of actions. If 0.1, 1.0 or 10% of total genes in a typical plant are valid for herbicide target, a plant with 50,000 genes would provide about 50, 500, and 5,000 targets, respectively. However, only 11 herbicide targets have been identified and commercialized. The successful design of novel herbicides depends on careful consideration of a number of factors including target enzyme selections and validations, inhibitor designs, and the metabolic fates. Biochemical information can be used to identify enzymes which produce lethal phenotypes. The identification of a lethal target site is an important step to this approach. An examination of the characteristics of known targets provides of crucial insight as to the definition of a lethal target. Recently, antisense RNA suppression of an enzyme translation has been used to determine the genes required for toxicity and offers a strategy for identifying lethal target sites. After the identification of a lethal target, detailed knowledge such as the enzyme kinetics and the protein structure may be used to design potent inhibitors. Various types of inhibitors may be designed for a given enzyme. Strategies for the selection of new enzyme targets giving the desired physiological response upon partial inhibition include identification of chemical leads, lethal mutants and the use of antisense technology. Enzyme inhibitors having agrochemical utility can be categorized into six major groups: ground-state analogues, group specific reagents, affinity labels, suicide substrates, reaction intermediate analogues, and extraneous site inhibitors. In this review, examples of each category, and their advantages and disadvantages, will be discussed. The target identification and construction of a potent inhibitor, in itself, may not lead to develop an effective herbicide. The desired in vivo activity, uptake and translocation, and metabolism of the inhibitor should be studied in detail to assess the full potential of the target. Strategies for delivery of the compound to the target enzyme and avoidance of premature detoxification may include a proherbicidal approach, especially when inhibitors are highly charged or when selective detoxification or activation can be exploited. Utilization of differences in detoxification or activation between weeds and crops may lead to enhance selectivity. Without a full appreciation of each of these facets of herbicide design, the chances for success with the target or enzyme-driven approach are reduced.