Soichiro Arai

Radioresistant DNA synthesis in fibroblast cell lines derived from LEC strain rats

Immortalized cell lines from LEC strain (LEC) rats by SV40 large T antigen were more sensitive to X-irradiation than the cell lines from WKAH strain (WKAH) rats. The dose-response curves for inhibition of DNA synthesis in WKAH-derived cells showed a steep decline at low doses and a shallow decline at high doses. On the contrary, the dose-response curves for LEC-derived cell lines showed no steep component; they were almost identical to the shallow component of the curves for WKAH-derived cells.

HIGHER SENSITIVITY IN INDUCTION OF APOPTOSIS IN FIBROBLAST CELL LINES DERIVED FROM LEC STRAIN RATS TO UV-IRRADIATION

The proportion of S-phase cells in a WKAH rat cell population decreased and that of G1-phase cells increased at 8 and 18 hr post-incubation following UV-irradiation, although no significant change was observed in the ratio of the proportion of S-phase to G1-phase cells in a LEC rat cell population. Thus, UV-radiation-induced delay in the progression from the G1 to S phase was observed in WKAH rat cells but was not apparent in LEC rat cells. The fraction of LEC rat cells containing a sub-G0 DNA content increased with post-incubation time after UV-irradiation, but not that of WKAH rat cells. The proportion of the sub-G0 fraction in LEC rat cells increased with increasing doses of UV-rays. Low molecular weight DNAs extracted from UV-irradiated LEC rat cells exhibited an intense DNA ladder pattern at 18 and 24 hr post-irradiation by electrophoretic analysis, but not those from UV-irradiated WKAH rat cells. These results showed a higher sensitivity of LEC rat cells in induction of apoptosis than that of WKAH rat cells to UV-irradiation, although there was no difference in the survival curves among the cell lines from LEC and WKAH rats after UV-irradiation.

Intramuscular Injection of Plasmid DNA Expressing mRNA7 Coding the Nucleocapsid Protein of JHMV Partially Protected Mice Against Acute Infection with JHMV

We constructed a plasmid expressing mRNA7 coding the nucleocapsid (N) protein of JHM strain of mouse hepatitis virus (JHMV) under the control of Rous sarcoma virus LTR, referred to as pRSV-mRNA7. When C57BL/6 mice injected intramuscularly (i.m.) with control plasmid DNA which contained no viral sequence were infected with JHMV, necrotic figures of neural cells and diffuse immersion of lymphatic cells in the pedunculus cerebri were observed. In the hypothalamus, vascular cuffing consisting of lymphatic cells was observed. In contrast, no histological change was observed throughout these areas of the brains in the JHMV-infected mice after injection with pRSV-mRNA7. These results showed that the injection with plasmid DNA expressing mRNA7 of JHMV partially protected mice against acute infection with JHMV in the brain. The plasmid DNA was i.m. injected into mice and the cytolytic activity of spleen cells from the mice was assessed by 51Cr-release assay. The spleen cells from the mice administrated with pRSV-mRNA7 showed a significant level of cytolytic activities against the transfected cells expressing the viral N protein even though the spleen cells were not cocultivated with stimulator cells. When the spleen cells from administrated mice with pRSV-mRNA7 were cocultivated with stimulator cells, higher cytolytic activity was observed against the transfected cells, compared with the activity without stimulation.

Inhibitory Effects of Modified Oligonucleotides Complementary to the Leader RNA on the Multiplication of Mouse Hepatitis Virus

Phosphorothioate oligonucleotides (PS-oligo) and PS-oligos with cholesterol conjugates (ChPS-oligo) complementary to the leader RNA of strain JHM of mouse hepatitis virus (JHMV) were more effective inhibitors of viral multiplication than natural oligodeoxynucleotides (PO-oligo) in JHMV-infected DBT cells. PS- and ChPS-oligos were 1,000 times more potent than unmodified PO-oligo. No significant difference was observed in the inhibitory efficiency between PS-oligo and ChPS-oligo. Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 M) of antisense PS-oligo and ChPS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20, and PS-(dC)20 with cholesterol conjugates, and PS- and ChPS-oligo which have no significant homology to the JHMV sequences, showed inhibitory effects on JHMV multiplication at concentrations higher than 0.5 M. These results showed that PS-oligo and ChPS-oligo were more potent than PO-oligo in the inhibition of JHMV multiplication, and that PS-oligo and ChPS-oligo may inhibit JHMV multiplication by two different mechanisms, that is by sequence-dependent and sequence-independent manners.