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Two core technologies, Laser Micro-beam Microdissection (LMM) and genome-wide cDNA microarray containing more than 32,000 transcripts, have facilitated specific and comprehensive gene expression profile analyses of various human tumors and normal tissues. Together with technologies of functional assays in OTS, we successfully identified a number of candidate molecules that will contribute to the development of novel treatment and/or diagnosis of human cancers.
Laser Micro-beam Microdissection (LMM)
Human cancer tissues consist of both cancer cells and non-cancerous cells including stromal cells and infiltrating lymphocytes as shown in the figure below. Therefore expression profile using bulk tissues does not reflect accurately profile of pure cancer cells. LMM enables us to obtain pure population of cancer cells from clinical cancer tissues. Although this technology requires an enormous amount of work to obtain a sufficient amount of RNA, we used this technology and obtained accurate and detailed expression profiles of cancer cells.
Genome-wide cDNA microarray
We established the genome-wide cDNA microarray system to obtain expression profiles. Our cDNA microarray contains more than 32,000 cDNA sequences that were prepared by RT-PCR using primer sets specific to each of the cDNA sequences. The primer sets were designed to amplify a single copy part of cDNA (200 and 1000 base pairs). Therefore, we can obtain the expression profiles with much less background and higher sensitivity than other microarray platforms. The combination of LMM and our microarray system enables us provide high quality and reliable expression profile data.
Molecular target genes
We obtained gene expression profiles of human tumor cells using LMM and our cDNA microarray as the first step. We also examined expression profiles of 30 normal human tissues including heart, liver, lung, kidney, placenta, ovary and testis as a second step. A comparison of the expression profile data of cancer cells with those of normal human tissues, we identified genes whose expression was abundant in cancer cells and absent or hardly detectable in non-cancerous cells. Through this approach, we also found many gene products that were considered to be cancer-testis antigens.
We then applied the RNA interference technology to examine whether each of the over-expressed genes was essential for the growth or survival of cancer cells. We further selected genes whose siRNA inhibited the growth of cells in which a target gene was highly expressed, but did not affect the growth of cells in which a target gene is not expressed. This multi-step analysis identified promising target molecules for the development of anticancer drugs or immunotherapy.
To clarify the function of the candidate molecules, we have been performing further biological analysis and have proven that some of these molecules have enzymatic activity, modulate signal transduction, and effect on cell cycle progression. For some of them, we identified their interacting proteins that are considered to be druggable targets.
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