Ex vivo simulation of drug action: quantification of drug-induced mRNA as a bridge between preclinical and clinical trials.
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Biomedical subjects
Masato Mitsuhashi
Publications and source records attributed to Masato Mitsuhashi.
Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis.
BACKGROUND: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. METHODS: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. RESULTS: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited approximately 10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%-40% were detected with statistical significance, and the experimental CVs were low (10%-20%). CONCLUSION: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression-based molecular diagnostics.
Adult↗
Application of differential display to identify genes for lung cancer detection in peripheral blood.
A blood assay for detection of lung cancer biomarkers could significantly improve cancer patient prognosis and survival rates. Amplified fragment length polymorphism-differential display (AFLP-DD) was used to identify gene transcripts found in lung cancer tissue and the peripheral blood of lung cancer patients. The clones were evaluated for gene expression in lung cancer tissue, peripheral blood of lung cancer patients and healthy volunteers' blood. The isolated gene transcript clones were found to be from the syndecan 1 gene, collagen 1 gene and 2 novel genes. All 4 transcripts were expressed in normal lung tissue, 4 cultured primary lung cells and 6 lung cancer cell lines. RNA was isolated from peripheral blood samples of 69 lung cancer patients. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to test for the presence of cytokeratin 19 and the 4 gene mRNA transcripts in blood RNA. The positive detection rate of at least 1 of the 5 transcripts was 79% for lung adenocarcinoma and 62% for squamous carcinoma. Using RT-PCR, at least 1 of the markers was found in 53% of stage I patients, 100% of stage II, 71% of stage III and 81% of stage IV lung cancer patients. Blood samples from 20 healthy volunteers were also tested, but only 1 of the 5 transcripts was found in 1 patient. These new molecular markers may aid early detection, staging and follow-up of lung cancer patients by RNA isolated from blood.
Adult↗
VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice.
The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)-transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 microg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO-treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO-treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.
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