Osamu Nikaido

The detection of cyclobutane thymine dimers, (6-4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects

Ultraviolet irradiation of skin induces various DNA photolesions. Here we demonstrate that irradiation of human buttock skin with 300 nm UVR in situ induces thymine dimers and 6-4 photoproducts. Irradiation with 260 nm immediately followed by UVA (320 nm) induces the Dewar photoisomers of the 6-4 lesions. All three lesions can be detected in methanol-fixed paraffin sections using specific monoclonal antibodies. The sections have been analysed in an automated image analysis system (Discovery) and the level of immuno-DAB-peroxidase measured in individual epidermal cell nuclei as absorption at 460 nm (integrated optical density). The staining patterns with the antibodies showed no detectable change with epidermal depth by eye after 300 nm irradiation, however, the machine detected a fall off with depth of about 2.5% per cell layer. Following irradiation with a shorter wavelength (260 nm) there was a rapid fall off in staining with depth easily detectable by eye and machine (39% per cell layer).

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

Abstract— By using specific monoclonal antibodies in situ and a computer‐assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad‐spectrum UVB and narrow‐spectrum UVB. The lamps produced these lesions in different proportions, with broad‐spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow‐spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad‐ or narrow‐spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad‐spectrum or narrow‐spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T‐lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T‐lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

Removal of UV-induced DNA lesions in mouse epidermis soon after irradiation

Induction and removal of cyclobutane thymine dimers and (6-4)photoproducts were studied in epidermal DNA isolated from UV-exposed hairless mice. For the detection of DNA damage, lesion-specific monoclonal antibodies were used in an immunoslotblot assay. Following the exposure of mice to 3.0 kJ m-2 UV-B, substantial removal of both thymine dimers (66%) and (6-4)photoproducts (77%) was observed at 24 h after irradiation. No removal, however, was detected at 4 h after irradiation. In contrast, immunofluorescence data obtained previously showed a rapid initial dimer removal after irradiation with 1.0 kJ m-2 UV-B (A.A. Vink, R.J.W. Berg, F.R. De Gruijl, L. Roza and R.A. Baan, Carcinogenesis, 12 (1991) 861-864). Reinvestigation of the removal of dimers and (6-4)-photoproducts shortly after three different UV doses showed a rapid decreases of both lesions at 2 h after irradiation with 1.0 kJ m-2. The results obtained after irradiations with 2.0 and 3.0 kJ m-2 UV-B suggest a saturation of repair already at 2.0 kJ m-2. Cyclobutane dimers were found to be removed at a lower rate than (6-4)photoproducts.

Detection of ultraviolet photoproducts in mouse skin exposed to natural sunlight.

In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine‐pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid‐summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV‐photoproducts in formalin‐fixed, paraffin‐embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time‐dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun‐exposed areas of human skin.

Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A.

US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US31 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A.

A Specific Chromosome Change and distinctive Transforming Genes Are Necessary for Malignant Progression of Spontaneous Transformation in Cultured Chinese Hamster Embryo Cells

Chinese hamster embryo (CHE) cell strains, each initiated from a separate cell stock obtained from different mothers, were transferred successively at intervals of 3 days and the changes in growth properties and karyotypes at various passages were examined. All nine cell strains proliferated at varying growth rates for 60 passages but only 2 (designated CHE A1 and CHE A2) of them expressed malignant phenotypes. The acquisition of tumorigenicity in nude mice was observed in CHE A1 and CHE A2 cells at passages 40 and 10, respectively. After 5 passages, 8 of 9 cell strains contained one or two common additional chromosomes, chromosome 3q and/or chromosome 5, although one cell strain (designated CHE A3) maintained a normal diploid karyotype for 60 passages. Trisomy of chromosome 3q was observed in all tumorigenic CHE A1 and A2 cells. One or two 3q chromosomes were detected in all tumor‐derived cell lines established from tumors produced by these tumorigenic cells. DNA from tumorigenic cells and tumor‐derived cell lines exhibited a high ability to transform mouse NIH3T3 cells, but we could not detect any activation of Ha‐ras, Ki‐ras, hst, erbB‐2, mos, met or raf in any of the transformed NIH3T3 cells. These results suggest that even though cultured CHE cells can transform spontaneously, without any specific chromosome change, to immortal cells, activation of unknown oncogene(s) in addition to a specific chromosome change may be required for their malignant progression. Our results suggest that trisomy of chromosome 3q is this specific chromosome change.