34. Studies Of Cellular And Molecular Mechanisms Of High LET Irradiations

Hideyuki Majima, Masao Suzuki, Kumie Nojima, Shizuko Kakinuma and Kazunobu Fujitaka

Keywords: MnSOD, promoter, DMSO, protection, PCC, mutation, LET, RBE

1) MnSOD promoter mutations cause gene disfunction
Manganese superoxide dismutase (MnSOD) has been shown to play an important role in preventing the development of cancer. MnSOD activity is reduced in many transformed cells and tumor tissues. We previously showed that the reduced level of MnSOD activity in cancer cells was not due to a defect in the primary structure of MnSOD protein, but rather was due to defects in gene expression. To elucidate the cause for the reduced expression of human MnSOD in cancer, we investigated the nucleoside sequence in the regulatory region of the MnSOD gene in a normal human cell line and various human tumor cell lines. A DNA sequence analysis identified three heterozygous mutations in the proximal region of the promoter in five human tumor cell lines. These mutations, clustered around the G C-rich region of the human MnSOD promoter, change the binding pattern of AP-2 and lead to a reduction in transcription activity using a luciferase reporter assay system. These results suggest that the reduced level of MnSOD expression in some tumor cells is, at least in part, due to a defect in the DNA sequence of the promoter region.

2) Effects of heavy particle irradiation on frozen mammalian cells.
We examined the effect of DMSO on sensitivity to carbon ions in this study. We tested cell growth and colony forming ability of cells that were kept frozen in new made Teflon dishes. We examined the effect of 10 % DMSO on colony ability after radiation by carbon ions of L5178Y and M10 cells. DMSO protected colonizing ability of two cell types.

3)LET dependence for cell death, mutation induction and chromatin damage when irradiated with different kinds of ion beams
We have been studying the LET dependence for cell death, mutation induction and chromatin damage in human cell lines irradiated with different kinds of ion beams. The effect of cell death was detected by colony forming assay. The mutation induction for hprt locus was investigated to measure the 6-TG resistant colony forming ability. The induction of residual chromatin breaks was measured by counting the number of remaining chromatin fragments after a 24-hour post-irradiation incubation, using the premature chromosome condensation (PCC) technique. The results for cell death showed that the RBE values, relative to 200kV X-rays, using 290MeV/n carbon ion beams were 1.44 for 20keV/m, 1.71 for 40keV/m, 2.14 for 60keV/m, 2.69 for 90keV/m, 2.48 for 101keV/m, 2.81 for 124keV/m and 1.11 for 217keV/m. The induction for mutant clones and residual chromatin breaks depended on LET values; RBE values increased with increasing beam LET values, when using 290MeV/n carbon ion beams (13, 40, 77 101keV/m). Both mutation induction and residual chromatin- breaks by carbon ion beams were 1.32 to 8.30 times higher than those by 200 kV X-rays.

Publications:
Majima H., Oberley T., Furukawa K., Mattson M., Yen H-C., Szweda L. and St.Clair D.:J.Biol.Chem., 273, 8217-8224, 1998.