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-24- Photobiology(2) 9/4 Room-B
VARIOUS ROUTES IN THE PRODUCTION OF H2O2 DURING UVA-IRRADIATION OF RIBOFLAVIN.
Kenji Sato1, Hironori Minami1,2, Takuo Tsuji1.
Departments of Dermatology, 1Nagoya City University Medical School, Nagoya, 2Osaka University School of Medicine, Osaka, Japan.
Flavins are a possible important chromophore for photo-induced skin injury. The primary in vitro cytotoxicity in riboflavin (Rf) solution irradiated with UVA is derived from hydrogen peroxide (H2O2). The irradiation of Rf solution with UVA in the presence of sodium azide (NaN3; singlet oxygen quencher) made the production of H2O2 smaller in volume. When excess NaN3 was present in the irradiation, the production of H2O2 decreased to 1/10 of the amount produced in the absence of the chemical. The measurement of oxygen consumption during UVA irradiation of Rf under nitrogen gas revealed that only half of oxygen atoms of the H2O2 produced derived from dissolved oxygen in Rf solution. These results indicate that singlet oxygen is involved partly in producing H2O2 in UVA-irradiated Rf solution and singlet oxygen- and oxygen-independent routes may be present in the production.
-25- Photobiology(2) 9/4 Room-B
THREE-DIMENSIONAL ANALYSIS OF NUCLEAR LOCALIZATION OF REPAIR SITES OF UV-INDUCED DNA DAMAGE IN HUMAN CELLS.
Akemi Nakagawa1, Nobuhiko Kobayasi1, Yukio Yamashina1, Tsutomu Muramatsu1, Toshihiko Shirai1, Toshio Mori2.
1Department of Dermatology, 2Radioisotope Center, Nara Medical University, Nara, Japan.
The two major lesions produced by UV are cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4PP). Both are repaired in normal human cells by nucleotide excision repair (NER). Despite the accumulated knowledge of NER, little is known about where they are repaired. To investigate this, normal human and XP-C cells were UV-irradiated, and incubated for various times in growth medium or BrdU-containing medium. The CPDs, 6-4PPs and BrdU in cellular nuclei were three-dimensionally visualized by laser scanning confocal microscopy with damage-specific antibodies (TDM-2; 64M-2) and anti-BrdU antibodies. The CPDs and 6-4PPs in DNA were quantitated by ELISA. BrdU incorporated into DNA during the repair period was determined by the quantitative immunofluorescence method. Normal human cells repaired 90% of initial 6-4PP within 3h, and removed 50% of initial CPDs at 24h after UV, respectively. They performed more efficient BrdU-uptake during the first 3h repair period than during the second 3h period. No significant removal of photolesions and unscheduled DNA synthesis (UDS) were seen within 24h after UV-irradiation in XP-C cells. We succeeded in three-dimensional visualization of CPDs, 6-4PPs, DNA repair synthesis and their corresponding nuclei in a single-cell level in UV-irradiated human cells. The punctate, not diffusely spread, nuclear localization of unrepaired 6-4PPs was found at 2h incubation. The similar nuclear localization of UDS was also seen at 3h incubation. The present results suggest that 6-4PPs and CPDs are non-randomly repaired from nuclei using the global genome repair subpathway in NER.
-26- Photobiology(2) 9/4 Room-B
WHAT IS XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP E (XP-E)?-BIOCHEMICAL ANALYSES OF XP-E CELLS.
Toshiki Itoh1, Tomomichi Ono2, and Masaru Yamaizumi1.
1Department of Cell Genetics, Institute of Embryology and Genetics, 2Dermatology, Kumamoto University School of Medicine, Kumamoto, Japan
Patients with xeroderma pigmentosum complementation group E (XP-E) have only mild clinical manifestations and show highest unscheduled DNA synthesis (UDS) among XP groups A-G. Recent study showed that XP-E had a biochemical heterogeniety in binding to UV-irradiated DNA. Some XP-E individulas lacked a damage-specific DNA-binding (DDB) activity in nuclear extracts. We report here the results of biochemical analyses using previously reported XP-E cells.
-27- Photobiology(2) 9/4 Room-B
CHARACTERIZATION OF MOLECULAR DEFECTS IN XERODERMA PIGMENTOSUM GROUP F.
Yasuhiro Matsumura1, Chikako Nishigori1, Sada Imamura1, HirakuTakebe2.
1Department of Dermatology, 2Department of Radiation Genetic, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
XP-F is characterized by mild clinical features in spite of low level of UDS. 20 patients have been reported so far, and all except three are Japanese. The cDNA that complements the repair deficiency of XP-F cell has been isolated recently. Here we characterized XPF mRNA mutations and examined the expressions of the mRNA and protein in seven primary cell strains from XP-F patients. Twelve types of alterations were found in the region between 642th and 1937th nucleotide and most strains were compound heterozygote. (1)XP3YO, XP7KA and XP101OS had two kinds of amino acid-substituted XPF proteins, (2)XP2YO and XP24KY had amino acid-substituted and truncated XPF paroteins (3)Only two strains, XP23OS and XP1TS had homozygous mutations which produce truncated XPF protein. The correlation between the site of gene alterations and the severity of clinical symptoms could not be observed. XPF cells express normal level of XPF mRNA, however, XPF protein could barely be detected, which suggested that the detected mutations would lead to unstable XPF protein expression, followed by the decrease in the amount of ERCC1-XPF endonuclease complex.( This work has been done by the collaboration with Dr.Yagi, Kyoto University)
-28- Photobiology(2) 9/4 Room-B
induction mechanism of uvc-induced hsp 72 in scc cell line.
Sayoko Hirai, Tsutomu Muramatsu, Toshihiko Shirai.
Department of Dermatology,Nara Medical University,Nara,Japan.
GST fusion proteins (FPs) were generated to five regions of the pemphigus vulgaris (PV) antigen. FP1, FP2 and FP3 were FPs containing the extracellular calcium binding sites. FP4 and FP5 were FPs encoding the non-calcium binding sites of the extracellular domainand intracellular domain, respectively. With the immunoblot analysis, we examined the sera from the patients with PV (n=20), pemphigus foliaceus (PF, n=12), paraneoplastic pemphigus (PNP, n=2) and bullous pemphigoid (BP, n=2), and 4 normal (NM) sera. Fourteen out of 20 PV seara reacted with these FPs. In contrast, 2 PF sera reacted only with FP1,and PNP,BP and NM sera showed no positive reaction.
-29- Photobiology(2) 9/4 Room-B
UVB-INDUCED MITOCHONDRIAL DYSFUNCTION IS ASSOCIATED WITH DECREASED CELL DETACHMENT OF CORNEAL EPITHELIAL CELLS IN VITRO.
Shigeto Shimmura, Eiki Goto, Kazuo Tsubota.
Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.
Purpose:To evaluate the effects of UV-B on mitochondrial function, cell viability and migration of cultured human corneal epithelial cells. Methods:Mitochondrial function following UV-B exposure in human corneal epithelial cells (T-HCEC) was assessed using rhodamine 123 (Rh 123). The oxygen consumption rate was measured by a Clark type O2 meter, and ATP contents were measured by chemoluminescence. Cell viability and migration was observed by propidium iodide (PI) staining and migration assays. Results:UV-B exposure caused a drop in O2 consumption by T-HCEC, while exposure of a mono-layer culture of T-HCEC to UV-B at 50 mJ/cm2 caused a reversible decrease in Rh 123 fluorescence (22.4 %), and a significant decrease in ATP contents (1.52 ÅÚ 0.05 nmol/106 cells) compared to control (2.93 ÅÚ 0.12 nm/106 cells). The effects on Rh 123 fluorescence was irreversible at 100 mJ/cm2, which approximately corresponded with the threshold dose at which cells positive to PI staining (PI (+)) appeared. UV-B doses of 50 mJ/cm^2 caused detachment of T-HCEC which were mostly PI (-), while higher doses (100 mJ/cm2) resulted in PI (+) cells that did not detach from the dish. Conclusions: UV-B (100 mJ/cm2) is associated with both irreversible mitochondrial dysfunction and with the loss of the ability for cultured corneal epithelial cells to detach in vitro.