To answer the question whether iron accumulation in transgenic rice seeds depends on the expression level of exogenous soybean ferritin, we generated two kinds of ferritin hyper-expressing rice lines by introducing soybean ferritin SoyferH-1 gene under the control of the rice seed storage glutelin gene promoter, GluB-1 and the rice seed storage globulin gene promoter, Glb-1, (GluB-1/SoyferH-1 and Glb-1/SoyferH-1, DF lines), and by introducing the SoyferH-1 gene under the control of Glb-1 promoter alone (Glb-1/SoyferH-1, OF lines). Ferritin expression was restricted to the endosperm in both lines and protein levels determined by western blot analysis were up to 13-fold higher than in a construct previously reported FK22 (GluB-1/SoyferH-1, in genetically Kitaake background); [corrected] however, the maximum iron concentrations in seeds of both of the new lines were only about 30% higher than FK22. The maximum iron concentration in the OF and DF lines was about threefold higher than in the non-transformant. The mean Fe concentration in leaves of ferritin over-expressing lines decreased to less than half of the non-transformant while that the plant biomasses and seed yields of the ferritin-transformed lines were not significantly different from those of the non-transformant, suggesting that accumulation of Fe in seeds of hyper-expression ferritin rice did not always depend on the expression level of exogenous ferritin but may have been limited by Fe uptake and transport. No obvious differences were observed for other divalent-metal concentrations (Ca, Cd, Cu, Mg, Mn and Zn) in the seeds among all experimental lines and non-transformant.

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5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein. [Cancer Res 2008;68(17):7035-41]

Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome-wide correlation analysis between DNA copy number and antitumor activity against 5-fluorouracil (5-FU)-based drugs (S-1, tegafur + uracil [UFT], 5′ ′ ′ ′-DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nuceotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5-FU-based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5-FU-based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response. (Cancer Sci 2007; 98: 577-583)

The CpG island methylator phenotype (CIMP þ ) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP þ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP þ was associated with a specific mRNA expression pattern for folate-and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP þ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP þ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of g-glutamyl hydrolase (GGH) was strongly associated with CIMP þ and CIMP þ -related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP þ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP þ tumours and could not account for the low expression level of this gene. CIMP þ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP þ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/ leucovorin.

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OPRT polymorphism predicts toxicity, especially grade 3 or greater diarrhea to oral UFT + LV adjuvant chemotherapy, whereas TS does not, in our study cohort. UGT1A1 polymorphism seems to be a risk factor for hyperbilirubinemia due to UFT+LV.

The development of a diagnostic method for predicting the therapeutic efficacy or toxicity of anticancer drugs is a critical issue. We carried out a gene expression analysis to identify genes whose expression profiles were correlated with the sensitivity of