Amplification of 4'-thioDNA and its utility as a shRNA expression system.

In this presentation, we discuss PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside triphosphate (sdNTPs). The amplified 4'-thioDNA acted as templates for not only in vitro transcription by T7 RNA polymerase, but also transcription in cells by RNA polymerase III. Accordingly, we succeeded to inhibit gene expression in cells by transfection of 4'-thioDNA that expresses a short-hairpin RNA (shRNA).

Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4'-thioribonucleosides.

A detailed study of the modification pattern-RNAi activity relationships by using siRNAs that are modified with 4'-thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa-2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4'-thioribonucleoside unit on both ends of the sense strand and four residues on the 3'-end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4'-thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.

In vitro simulation study of individualized chemotherapy in lung cancer.

The primary aim of this in vitro simulation study was to evaluate the utility of gene expression profile analysis in predicting the effect of varying drug combinations for the treatment of lung cancer. Using 10 human cancer cell lines, we focused our gene expression analysis on a cohort of candidate sensitivity-prediction factors, previously reported using cDNA filter arrays, with a view to predicting the ability of a set of anti-cancer drugs commonly used to treat lung cancer, namely cisplatin, 5-fluorouracil (5FU), SN38, docetaxel, gemcitabine, and vinorelbine. Altered expression of genes for glutathione-S-transferase-pi, uridine phosphorylase, O-6-methylguanine-DNA methyltransferase, and multidrug resistance 1 was identified in lung cancer cell lines. Drug sensitivity testing, in the form of methylthiotetrazol analysis, was performed using these six anti-cancer drugs against the panel of 10 lung cancer cell lines. We compared the predicted chemosensitivity based on the gene expression pattern of 19 well-known sensitivity-related genes with the cytotoxic activity of each of these anti-cancer drugs. Molecular profiling data predicted resistance to CDDP in LK-2 cells, 5FU in LK-2, PC7, A549, NCI-N231, Lu135 cells, irinitecan in PC9 cells, and VNR in PC7 cells. However, the prediction efficacy (number of predicted inactive drugs by gene expression analysis/number of inactive drugs by methylthiotetrazol assay) was 21.6% (8 of 37). No false-positive findings in relation to sensitivity-related genes were obtained on the basis of this molecular analysis. Thus, prediction of sensitivity to lung cancer by molecular analysis appears possible. With elucidation of additional drug sensitivity factors, selection of appropriate anticancer drugs by gene expression profiling may make it possible to increase the response rate in lung cancer chemotherapy.

Anticancer drug clustering in lung cancer based on gene expression profiles and sensitivity database.

BACKGROUND: The effect of current therapies in improving the survival of lung cancer patients remains far from satisfactory. It is consequently desirable to find more appropriate therapeutic opportunities based on informed insights. A molecular pharmacological analysis was undertaken to design an improved chemotherapeutic strategy for advanced lung cancer. METHODS: We related the cytotoxic activity of each of commonly used anti-cancer agents (docetaxel, paclitaxel, gemcitabine, vinorelbine, 5-FU, SN38, cisplatin (CDDP), and carboplatin (CBDCA)) to corresponding expression pattern in each of the cell lines using a modified NCI program. RESULTS: We performed gene expression analysis in lung cancer cell lines using cDNA filter and high-density oligonucleotide arrays. We also examined the sensitivity of these cell lines to these drugs via MTT assay. To obtain our reproducible gene-drug sensitivity correlation data, we separately analyzed two sets of lung cancer cell lines, namely 10 and 19. In our gene-drug correlation analyses, gemcitabine consistently belonged to an isolated cluster in a reproducible fashion. On the other hand, docetaxel, paclitaxel, 5-FU, SN-38, CBDCA and CDDP were gathered together into one large cluster. CONCLUSION: These results suggest that chemotherapy regimens including gemcitabine should be evaluated in second-line chemotherapy in cases where the first-line chemotherapy did not include this drug. Gene expression-drug sensitivity correlations, as provided by the NCI program, may yield improved therapeutic options for treatment of specific tumor types.

14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for Miller-Dieker syndrome.

Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3epsilon (YWHAE), one of a family of ubiquitous phosphoserine/threonine-binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3epsilon results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3epsilon in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.