遺伝子発現を利用した照射効果測定

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The effect of X-irradiation on production of MUC1 was studied with human colon carcinoma HT-29 cells. Monoclonal antibody (mAb) MY.1E12 was used to identify MUC1. Immunomicroscopically, the percentage of MUC1 positive cell was 52% in 6 Gy irradiated cells at day 4, compared with 26% in unirradiated cells. By flow cytometric analysis, MUC1 on 6 Gy irradiated cells was elevated at day 1, reaching plateau by day 4. Westerm blot analysis showed both 500 kDa and 390 kDa bands corresponding to two polymorphic MUC1 alleles were increased after X-irradiation. The transcriptional activity of the MUC1 gene was analyzed by transient expression of MUC1-CAT reporter plasmid containing the 5'-flanking sequence of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Elevation of CAT activity was observed in parallel with the expression of MUC1 after 6 Gy of X-irradiation. Moreover, to investigate in vivo effects, we developed a stable high-expression green fluorescent protein (GFP) transfectant of human colon carcinoma HT-29 cells. GFP-expressing cells (HT-29/GFP) were transplanted into the hind rimb of nude mice. After 40 days, tumor-bearing nude mice were irradiated of 0, 6, 10, 20, 30, 50 Gy. After 4 days, Westem blot analysis showed both 500 kDa and 390 kDa bands were increased of tumor derived HT-29/GFP cells after X-irradiation. These findings indicate that enhanced expression of MUC1 in irradiated HT-29 cells was due to upregulation of the MUC1 transcription.