025 Three-dimensional analysis of nuclear localization of repair sites of UV-induced DNA damage in human cells

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JSID Abstracts

025

028

THRBB-DIMENSIONAL ANALYSIS OF NUCLEAR LOCALIZATION OF REPAIR SlTES OF W-INDUCED DNA DAMAGE M HUMAN CELLS. & k4E4mwd &Iy&&&l~Y.~T.~l,,

INDUCTION MECHANISM OF UVC-INDUCED HSP 72 IN XC CELL LINE S.Hirai*, T.Muramatsu, TShirai. Department of Dermatology, Nara Medical

l Lleputmau of Dermsrology and 2 RI Center, Nsrs Medical University, Nars, Japan. The two mlpr lesiotu pmdwd by UV am cyclcbutane pyrlmkiine dimer (CPD) and (6-l)phMopmdu~t (6-W). Both are repaired in namsl human cells by nuclmlide excision repair @EFt). Despite the accumulated knowledge of NBB. liuk. is known shout where they are repaIred. To investigate.this. normal human and W-C! cells w.rc Wln’sdlaled, and incubrled for w&s times in growth medium or BrdUumtaining medium. The.CPDs. 64PPs and BrdU in ce4h1lsrnuclei were three-dimensionally vIawAked by lasu scanning confocal micq with damage-specific antibodies (TDM-2: 64M-2) and anti-BW antibodies. The CPDS and 6-4FTs in DNA were quantitated by ELlSA. BrdU incorporated into DNA during the repair period wss determined by the quantitative immunotlwescence. method. Normal human cells repaired 90% of initial 6-4FP within 3h. and removed 50% of Ihe initial CPDs st 24h afw W, res~ecdvely. They p&mud more efliiient BrdU-uptake during Ihe.first 3h repsk period lhan during the second 3h period. No signiliiant removal of photolesions and unscheduled DNA sywhesis (UDS) wcn seen wilhin 24h after W-imdiatim in XFC cells. We succeeded in duee.dimensional visuslizadon of CPDs. 64PPs. DNA repair synlheaiaand theii comsponding nuclei in a singlc-all level in UV-irradiated human cells. m punctale. not diffusely spsad, nuclear localization of unrepaired 6-4PPs was found st 2h incubsdon. The similar nuclear localization of UDS was also seen st 3h incubation. The present nsulrs suggest ti 64pW and CPDs sre non-randomly re@red frm nuclei using the globsl genome repair subpathway in NBR.

026 WHAT IS XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP E (XP-E)?-BIOCHEMICAL ANALYSES OF XP-E CELl.S._T. &&I% T. Onol. and M. Yamaizudll ‘Department of Cell Genetics, Institute of Embryology and Genetics, %rmatology, Kumamoto University School of Medicine, Kumamoto, Japan Patients with xemderma 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 UVirradiated DNA. Some XP-E individulas lacked a damage-specific DNAbinding (DDB) activity in nuclear extracts. We report here the results of biochemical analyses using previously reported XP-E cells.

027 CHARACTERIZATION OF MOLECULAR D;;ECTS IN XERODERMA PIGMENTOSUM GROUP F. Y. MatSumura ,C.Nlshlaon “2LJmammImamura’~ -2. Department of Dermatology’ and Department of Radiation Genetic, Graduate School of Medicine2, 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 is&ted recently. Here we characterized XPf mRN,& mutations and examined the exoressions of the mRNA and orotein in seven orimarv 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 )XPJYO. XP7KA and XPl 01 OS had two kinds of amino acid-substituted X@F’proteins, (2)XPZYO and XP24KY had amino acid-substituted and truncated XPF oaroteins (3)Onlv two strains. XP23OS and XPlTS had homozygous’mutations’wt&h broduce 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 mutat& would lead to &table XPF protein expression. followed by the decrease in the amount of ERCCl -XPF endonuclease complex.( This work has been done by the collaboration with Dr.Yagi, Kyoto University)

University,

Nara, Japan.

We examined

the binding

activity

of heat

shock

transcription factor (HSF) to the heat shock element (HSE) of the hsp72 gene promoter after UVC irradiation by a gel mobility-shift increased markedly Furthermore,the

assay. The activated after WC irradiation.

UVC-induced

HSP72 was suppressed

PKC inhibitor These results

(CAL) but not by PKA suggest that UVC-induced

accumulation

is

regulated

PKC might be involved

HSF level

by

by

inhibitor (H89). HSP72 transcriptional level and

in the activation

of HSF.

029 WB-INDUCED MITGCHONDRIAL DYSFUNCTION IS AS%XIATED WITH DECREASED CELLDETACHMENT OF CORNEZALEPITHE~ CELLSINVlTRO.ShlnetoShlmm~l Goto. ~Tsubota. Department of OphThalmoW, Tokyo Dental CoIlege. Chlba. Japan. Purpose:To evaluate the effects of W-B on mltochondrial function. ceU vlablllty and m&r&on of cultured human comealeplthellal cells. Methods:Mltochond function following W-B exposure In human comeal epltheIlal cells (T-HCEC) was assessed using rhcdamlne 123 (Rb 123). The oxygen consumption rate was measuredby a Clark OIpe 0s meter, and ATP contents were measured by chemolumlnescence. Cell vlablllty and migration was observed by wopldlum lodlde (PI) stalnlng and mlgratlon assays. Results: W-B exposure caused a drop In 0s consumption by T-HCEC, while exposure of a mono-layer culture of T-HCEC to UV-B at 50 mJ/cmZ caused a reversible decrease In Rh 123 fluorescence (22.4 96). anda slgnlflcant decrease In ATP contents (1.52 f_ 0.05 nmol/l06 cells) compared to control (2.93 f 0.12 nm/106 cells). The effects on Rh 123 fluorescencewas Irreversible at 100 ml/cm’, which approximately correspondedwith the threshold dose at which cells wsltlve to PI stalnlng (PI (+)) appeared. W-B doses of 50 mJ/cm-2 caused detachment of T-HCEC which were mostly PI (-). while higher doses (100 mI/cm’) resulted In PI (+) cells that did not detach from the dish. Conclusions: W-B (100 ml/cm’) Is associated with both lrreverslble mltochondrlal dysfunction and with the loss of the abIll& for cultured comeal eplthellal cells to detach In vitro.

030 INDUCTION

OF APOPTOSIS

PIG SKJN. M.Narrata. j%Yasuno. Department

BY HEAT

of Dermatology,

Kyoto

STRESS @fectual

IN GUINEA University

of

Medicine, Kyoto, Japan. Unwanted cells are eliminated by apoptosis, when cells are damaged. In this study, apoptosis was examined in the experimental burn wounds under some severities of heat stress by TUh’EL staining. Bum wounds, lcm in diameter, were made on thighs of guinea pigs by electorical imn for 5-M) min at 43’C. 5-60 min at 45’C. 60 min at 55-C. and 3 set at 18O’C. respectively. Skin samples were excised at day 2 after burn. Apoptosis was seen through epidermis to lower dermis in central region of the wound at 43’C, 5-20 min. Necrosis was seen in central region of the wound but apoptosis was seen in peripheral region of the wound at the other conditions. Intermediate damage (43’C, 5-20 min) induces apoptosis, that seems to be second burn, and more damage induces necrosis in guinea pig skin