Application of H.I.C.E.P., a novel method which can detect known and unknown gene expression changes after exposure to low dose x-rays

Although one can observe rapid advances in gene expression profiling method, it still is not easy to access the biological effects of radiation doses at the level of diagnostic radiology. In order to obtain better sensitivity, we used a novel gene expression profiling method, HiCEP (high coverage gene expression profiling )(Nucleic Acids Res. 31, e94) recently developed in our institute. HiCEP assay is an AFLP-based profiling method and has seven basic steps which include 1) double strand cDNA (dscDNA) synthesis with biotinylated oligo(dT) primer, 2) Digestion of dscDNA by Mspl (or Msel), 3) Ligation with Mspl (Msel) adapter and purification by magnetic beads coated with avidin, 4) Digestion o f dscDNA by Msel (or Mspl), 5) Ligation with Msel (or Mspl) adapter, 6) Selective PCR with 256 primers (16 X 16), 7) Detection of the fluorescence from selective PCR products. These products were analyzed by capillary electrophoresis. In data profile, the x-axis indicates the length of PCR products and the y-axis indicates the intensity of fluorescence of the PCR products. This assay is highly reproducible and sensitive enough to detect 1.2-fold difference in gene expression. Moreover, the low false positive rate enables us to analyze gene expression with wide coverage. It is estimated that 70-80% of all transcripts which include non-coding transcripts and unknown and known genes. Using HiCEP, we searched through over 23,000 transcripts in normal human fibroblasts (HFL III) irradiated with low dose X-rays. More than 200 genes are up-regulated transiently during one hour after 1 cGy X-rays. We determined the nucleotide sequences of 10 up-regulated transcripts with the greatest rate o f increase. Three out of these encoded a set of CXC chemokine genes (CXCL1, CXCL2, and CXCL6). The rest included the transcripts of other secretory product s and unknown genes. In order to test the involvement of CXC chemokines in cells irradiated with low doses, we irradiated HFL III cells with 1-10 cGy X-rays, and transferred the media from HFL III culture to two melanoma cell lines characteristic of excessive numbers of the CXC chemokine-specific receptors. The growth of these melanoma lines were significantly stimulated after the addition of HFL II I medium irradiated at 1-5 cGy (Cancer Res. 65, 10159-10163, 2005). These results suggest an existence of a defense mechanism specific for exposure to low dose s of ionizing radiation. (N.C.)