The DNA methylation machinery as a target for anticancer therapy

DNA methylation is now recognized as an important mechanism regulating different functions of the genome; gene expression, replication, and cancer. Different factors control the formation and maintenance of DNA methylation patterns. The level of activity of DNA methyltransferase (MeTase) is one factor. Recent data suggest that some oncogenic pathways can induce DNA MeTase expression, that DNA MeTase activity is elevated in cancer, and that inhibition of DNA MeTase can reverse the transformed state. What are the pharmacological consequences of our current understanding of DNA methylation patterns formation? This review will discuss the possibility that DNA MeTase inhibitors can serve as important pharmacological and therapeutic tools in cancer and other genetic diseases.     

Inhibition of Ras prenylation: a signaling target for novel anti-cancer drug design

This paper describes a measure of explained variation (MEV) of survival times for a given regression model used in survival analysis. It quantifies the predictive power of a set of prognostic factors in the model, and therefore provides useful information for more precise prediction of patient prognosis, and for designing randomized clinical trials with the capability of determining treatment effects. The MEV defined in this article is asymptotically derived from the squared product-moment correlation; it can be interpreted as an adaptation of the multiple correlation coefficient for the normal linear model to the survival time regression model. Monte-Carlo simulations are performed to investigate the statistical behavior of the proposed MEV. The MEV is applied to estimate the predictive power of several sets of prognostic factors for gastric cancer in Japan using data from a large clinical trial.     

Enzymatic DNA methylation as an aging mechanism

By analysis of 70 functional parameters of organism it has been shown that the senescence of human organs starts from the age of 20 +/- 10 years and it has more or less a linear character in the most cases. Judging by the averaged aging rate, human maximal lifespan can be equal to about 145 years. An aging model has been proposed according to it the functional involution is caused by cells destructing and is a result of gradual accumulation of special mutations in genome. The evidences has been obtained that enzymatic DNA methylation is a powerful generator for the 5mC-->T+C transition, constantly occurring with each cell division. It has been found that the age-related loss of the most part or all 5mC residues from DNA coincides with both the Hayflick limit in cell lines and the maximal lifespan of different animal species studied. The rate of DNA hypomethylation is proportional to the cell aging rate in vitro and in vivo. The 5mC-->T transitions contribute more than half of all point mutations, accumulated in genome of vertebrates during evolution. These substitutions occur ten times more often in the methylated sites than in other positions of DNA and they disproportional contribute to the general mutagenesis and to the hereditary human diseases. The conclusion has been made that DNA methylation meet the requirements of the main criterions of an aging biomarker and it can be considered as a mechanism for genetically programmed accumulation of mutations with aging. In immortal cell lines this mechanism starts working in the reverse direction, increasing the 5mC content in DNA.     

Choline kinase inhibitors as a novel approach for antiproliferative drug design

Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly specific inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with specific growth factors. A more profound inhibition of cell proliferation was observed in ras-, src- and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this effect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not affect ChoK activity. The inhibitory effect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.     

Drug target validation and identification of secondary drug target effects using DNA microarrays

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.     

组蛋白甲基化:肿瘤治疗的新靶标

暴露在核小体表面的N-末端尾部可发生共价修饰,这些共价修饰可以影响组蛋白和DNA结合的紧密程度,从而影响DNA的表达,被称之为"组蛋白密码".近来大量的研究表明,组蛋白末端的赖氨酸和精氨酸残基的异常甲基化与肿瘤的发生、发展、预后有着密切的关系.应用组蛋白甲基转移酶/去甲基转移酶抑制剂调控组蛋白的甲基化水平,能够抑制肿瘤细胞的生长并诱导凋亡.     

Lipoteichoic acid as a target for antimicrobial action

Fifteen patients with unilateral functioning arteriovenous fistula were assessed clinically and electromyographically to identify local neurologic changes. Ten of the patients were symptomatic (motor and/or sensory) and 5 were asymptomatic. Clinically, 11 patients had signs of a mild polyneuropathy, 2 patients of ulnar neuropathy, and 1 patient had signs of median neuropathy. A decrease of the above-elbow ulnar conduction velocity was noted in the study group on the side of the functional fistula, and in the symptomatic patients only on the side of the nonfunctional/nonexisting fistula. We suggest that ulnar nerve vulnerability should be taken into consideration during construction of the fistula, as well as during dialysis.     

p53 as a target for anti-cancer immunotherapy

The key to specific and non-toxic cancer therapy is likely to be identification and targeting of processes that are absolutely unique to the tumor. One such approach is to target cells expressing mutations in the oncoproteins that led to the development of the cancer, such as p53. In animal model systems, highly mutant p53-specific cytotoxic T cells can be induced, but it remains to be seen whether this can be translated into clinical practice, and what proportion of tumors will respond. In this review, the potential and problems of immunological targeting of mutant p53 in solid tumors are discussed.     

Topoisomerase II as a target for anticancer chemotherapy

Type II DNA topoisomerases are required for the segregation of genomic DNA at cell division in prokaryotic and eukaryotic cells, and inhibitors of these enzymes are potential cytotoxic agents in both prokaryotes and eukaryotes. The bacterial member of the topoisomerase II family, DNA gyrase, and the chemotherapeutic agents which target it are the subject of a recent review (Maxwell, A. et al., 1993, in Molecular Biology of DNA Topoisomerases, Andoh, T. et al., eds., pp. 21-30, CRC Press, Boca Raton). Here we present an overview of current knowledge of eukaryotic topoisomerase II and the anticancer agents which target this enzyme, focussing predominantly on new observations and recent reports and reviews.     

Evidence for gene silencing by endogenous DNA methylation

Transformed cells can spontaneously silence genes by de novo methylation, and it is generally assumed that this is due to DNA methyltransferase activity. We have tested the alternative hypothesis that gene silencing could be due to the uptake of 5-methyl-dCMP into DNA, via the di- and triphosphonucleotides. 5-Methyl-dCMP would be present in cells from the ongoing repair of DNA. We have isolated a strain of Chinese hamster ovary (CHO) cells, designated HAM-, which spontaneously silences two tested genes at a very high frequency. We have shown that this strain incorporates 5-[3H]methyldeoxycytidine into 5-methylcytosine and thymine in DNA. It also has low 5-methyl-dCMP deaminase activity. Another HAM+ strain has high deaminase activity and a very low frequency of gene silencing. The starting strain, CHO K1, has a phenotype intermediate between HAM- and HAM+.     

A PCR-based strategy for extensive mutagenesis of a target DNA sequence

A mixed population of mutagenic oligonucleotide primers was used to generate a set of point mutations in a short region of a retroviral gene by PCR amplification. The mixed population of mutagenic primers was generated by incorporating a mixture of A, G, C and T at specific sites during oligonucleotide synthesis. With the proportions of mutagenic nucleotides used for our experiments, 47 percent of the 213 clones analyzed had one or more point mutation in the target DNA sequence. In addition, unpredicted mutations were observed that contributed to the mutagenic complexity of the population. We have found this approach to be an efficient means for extensive mutagenesis of a defined target DNA sequence.     

Thymidylate synthase inhibition: a structure-based rationale for drug design

The effect of drugs in the fetus can be thought of in terms of the dose-response based on a knowledge of the general principles by which drug is transferred from the mother to the fetus. Fetal drug concentration, which determines the fetal response, is a function of the maternal concentration, the placental permeability, the fetal drug clearance, and differences in protein binding and ionization between the maternal and fetal plasma.     

Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer

The Y1 receptor, which belongs to the family of rhodopsin-like GTP-binding protein-coupled, seven-transmembrane helix-spanning receptors, binds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand-receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.