Zhen-Bo Han

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.

A genetic effect of altered gravity : mutations induced by simulated hypogravity and hypergravity in microsatellite sequences of human tumor cells

To determine the possible genetic effects of gravity alterations, we analyzed mutation induction in microsatellite sequences of human tumor cells treated with simulated hypogravity provided by a clinostat or hypergravity by a centrifuge. Microsatellite mutations were detected as changes in the size of polymerase chain reaction (PCR)-amplified allelic markers. The frequencies of mutant clones in cultures treated with simulated hypogravity for 24 or 48 h were almost the same as those of controls, but after 72 h of treatment, the mutant frequencies had increased significantly in all three microsatellite loci examined. Significantly higher mutant frequencies were similarly detected in cultures treated for 72 h with a hypergravity condition as low as 18xg, but not detected in 24 or 48 h treated cultures. These findings clearly show that gravity alterations that last for 3 days can induce microsatellite mutations in human cells. A genetic effect of gravity change, therefore, is established for the first time. Moreover, high frequencies of microsatellite mutations were induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C-mediated signal transduction pathways and causes genetic instability. These findings suggest that gravity change induces microsatellite mutations by modulating the pattern of gene expression involved in signal transduction pathways.

Neoplastic transformation of hamster embryo cells by heavy ions

We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/micrometer. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/micrometer, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.