Establishment of a human malignant endothelial cell line (ISO-HAS) from angiosarcoma arising on the scalp

ESDR I JSID I SID Abstracts

0352

0349 DIFFERENTIAL LOW

DOSES

p53-DEPENDENT OF

STRESS

ULTRAVIOLET

APOPTOSIS VLi. Division of Dermatology,

RESPONSE

RADIATION:

The University

Vii, of British

TO HIGH

AND

REPAIR

OR

DNA

Department of Medicme, Columbia, Vancouver, BC,

Canada ~53 is a crucial protein which plays an essential role in maintenance genetic stability afler a DNA damaging event, such as ultraviolet radiation

of the (WR).

After WR, the amount of p53 protein is elevated. The increased p53 is believed to induce cell cycle arrest, promote nucleotide excision repair (NER) and apoptosis. To study if cells respond differently to high and low doses of UVQ we examined DNA repair efficiency and apoptosis rate of human tibroblasts after WR. Using a host cell reactivation assay, we found that NER was increased after low doses but not high doses of WR.

The induction

of NER was only observed

in ~53”’ murine

tibroblasts, not in ~53” cells, indicating the induction of NER IS dependent on wild-type p53 function. p53-dependent apoptosis occurred after the cells received high doses (over 200 J/m*) of WB. WR induced the expression of ~53 protein in a dose-dependent manner. In contrast, p21’“‘““” was induced only at?cr low doses and bax only after high doses of WR, supporting the roles of ~21 and bax in NER and apoptosis, respectively. Taken together, these results indicate that cells respond differently apoptosis function.

depending atlcr high

on the dose of UVR: doses; both mechanisms

DNA repair at&r low doses arc dependent on wild-type

and ~53

WOUND DERMAL

FLUID

FROM CHRONIC

FlBROBLASTS.Martm

Park,Department

of Dermatology,

LEG LTLCERS GROWTH ARRESTS NEWBORN Leverkus, Tania 1. Phillips and Hee-Young

Boston University

School of Medicine,

Boston, MA

Chronic leg ulcers represent one of the major types of chronic wounds, mostly affecting elderlv oatients. It has been sueeested that the microenvironment of chronic wound. espe&liy the wound fluid, may -&dficantly contribute to the impairment of healing process. To explore whether wound fluid present in chronic leg ulcers (CWF) participates negatively in the healing process, CWF from leg ulcer patients, duration months to years, were collected bv aovlvine Allevvn dressine ovemieht and extractine the CWF usioe a 1 ml sterile syringe. CWF was then a&yed for & abili& to influence proliferation of &roblasts cultured from healthy human neonatal foreskin. Paired cultures of fibmblarts were treated L

with either CWF

1..

I

(500 u/ml)

or bovine serum albumin (BSA)

as a control

for 8 -

10 devs.

Cells were refed with f&h medium containing CWF on day 5. CWF samples collected from ten patients all inhibited growth of newborn tibroblasts by > 90%. Derroal fibroblasts play a crit&l role during woeid healing by providing matrix&, cytokines and growth &ors necessary for the regeneration and remodeling of dermal and epidermal tissues To examine if CWF causescells to growth arrest, paired cultures of newborn tibmblasts were treated

with either CWF or BSA for 24 - 36 hours, harvested and their growth state wes analyzed by FACS-Scan. Parallel culteres were treated with CWF or BSA and were also labeled with Brdu, a compound known to be incorporated into DNA only during DNA synthesis of cell cyle (S-phase), for 90 min. Both DNA contents and the intensity of Brdo indicated that 37% of the BSA-treated cells were in S-phax whereas < I% of CWF-treated cells were in Sohase. CWF-treated cells 0 50%) were mostlv in Ge (adescent)-obese. Initial column chromatography hss suggested that the factor(s) respo&ie for the inhibition of growth in

.

CWF

had a molecular weight lower than 100 kd. Combined together, our results suggest that CWF may participate in the impairment of wound he&g by growth are&g fibroblest which play e critical role in the healing process.

0350

0353

EVIDENCE FOR ACTIVE LTYROSlNB DE NOVO SYNTHESIS IN HUMAN EPIBERMAL MELANOCYTBS AND KBRA’IINOCYTES. W.D. Be&w. N.A. Hibberh. A. Shoulder. K.U. Sa Clinicaland Eqerimatel Dem~lfology, Dqertmat ofBiuazdicel Sciences, UnivemityofBmdford, UK

INCREASED MI-SOD EXPRESSION IN PSORIATIC FTBROBLASTS: MODULATION BY RETINOIC ACID. Franc&c Ravnaud. Pas&c Gerbaud. Patrice Therond.anie DimonGidal. Guv Kerver. Dani& Evain-Brian, Uniti INSERM 427, Facult6 de Pharmacie, Paris V. France. Psoriasis is a common, chronic, polygens. remitting and relapsing scaly and inflammatory skin disorder. We reported en abnormality m CAMP analog binding and a decreased CAMP-dependent protein kineses (PKAs) activity in psoriatic fibroblasts which can be reversed bv rctinoic acid. We showed that the alteration of PKAs is related to an oxidative mod&ion. This suggested a change in the oxidative state of psoriatic cells. Antioxidant enzyme activities were dctcrmined in normal fibroblasts and psoriatic fibmblasts. The most significant differences were noted in superoxide &smutase activities. A dramatic (5.2.fold) increase in Mn-SOD activity along with a lesser extend (LB-fold) incrcasc in CuZn-SOD was observed in psoriatic fibroblasts from lesional and nonlesional psoriatic skin compared to normal ones. The increase in Mn-SOD activity was associated with an increase in MnSOD pmtcin expression (western blot analysis). In immunocytochcmishy, using a specific palyclonal MnSOD Ab, the staining decorated the mitcchontias. An higher number (65%) of positive immunostaincd cells was observed in psoriatic flbmblasts as compared to normal ones (22%). Time course of rctinoic acid (1 @Q trcatmmt of psoriatic tibroblast revealed at 3 hours up to 4 days a significant (p=zO.Ol) dccrcasc in MnSOD activity in lesional (from man f SEM 242 f 6 to 49 f 7 mUI/mg prot) and nonlesional (220 t 22 to 34 f 2) skin; no effect was observed in normal tibmblasts (20 + 7 to I7 f 6). Retinoid action appears to be mediated through direct binding to nuclear receptms, however sequence analysis of MnSOD gene does not show any retinoic acid responsive element. Thcxfae this study suggests that rednoic acid acts as antioxidant in psoriatic tibroblasts, correcting the altered oxydativc state of psoriatic tibroblasts.

andfromu&f&&&~ken6nksatmocytesandmclen-~. Jnordmtomofmntbe mRNAexpreJJimtotalRNAwtuextradedfrcmceUcultunsandrweroetraarcribsdfdlowed by PCR using specific p&x8 for Qd key enzyme for 6BIi. de nm=u syldbesis, i.e. GTP cycl&ydrolase I (GTP-CI--I), GTP-CH-I feedback m&tory (GFBP). PAIi and the retc limiting recycling enzyme C-carbinotie dehydretase (Z-I) es well as Tm llu ms,dts demammted mRNA expreseim for all enzymes and for GF’BJ in eesocietia, witi, rapecteble enzyme activxties io b& melaeocytes and keretincqtes and in +lemml call extra&. Enryme activities are significantly lower in damniated cells compared to lmdab&&&pmlifbmtieg cells. Our data show for the first time that melmccyta end keretinw hold the 11l capeaty for active L-tymsine synulcsis from Lphenyialanine aud do act mly an active wnsport of Uds amino acid for intracellular L-tyrazine dependent processes suchBPcated,olsmine Jynmesis and pigmmtetian.

prasin

0351

0354 OF A HUMAN MALIGNANT ENDOTHELIAL CELL LINE (ISO-HAS) FROM ANGIOSARCOMA ARISING ONTHE SCALP. Hisamichi Hara, Mikio Masuzawa, Yuhko Hamada, Shiseo Nishivama and Kensei Katsuoka, Department of Dermatology, Kitasato University School of Medicine. Saaamihara. Japan. We have recently established a murine anglosarcoma cell line (ISOS-1). Using this culture conditioned medium in 50% concentration, we isolated and established a cell line (ISO-HAS) from tumor tissue of human angiosarcoma arising on the scalp. The cells retained endothelial cell properties such as a characteristic cobblestone appearance at confluence, contact-inhibited growth, and active uptake of acetylated low-density lipoprotein (Dil-AcLDL), although they were partially positive for von Willebrand factor antigen and blood type H antigen. They were also expressing several oncogenes (c-myc, c-fos, c-jun, p-53). The cell culture was maintained for more than one year up to 100 passages. These properties suggested that ISO-HAS was an endothelial cell line derived from angiosarcoma. Moreover, ISO-HAS expressed fatal tumorigenicity in viva, because the cells inoculated into SCID mice subcutaneously showed solid tumor growth that caused death. It may be a first report to establish a human malignant endothelial cell line. ESTABLISHMENT

We recently shown that ultraviolet.4 (WA) rapidly activates integrin expression in human de& tibmblasts grown in monolayer on plastic. In particular, U/cm2 of WA increases both the expression of at, a~, and o.5 chains at the cell surface, and the number of positive cells. In the present work, we invcstigatcd the influence of cell-matrix interactions existing at the time of irradiation, on focal adhesion kinase @AK) activation. Fibroblasts, firstly multiplicatcd in monolayer, were seeded according to two different conditions: in monolayer on plastic or in monolayer cm collagen type I coated Petri dishes. Cul~res were WA irradiated 3 days later (prccontluent culture) using a ViibcrtLouat lamp (ulcmz and toJlcm2). Immediately at& irradiation or sham-irradiation, tihmblasts were collected at 4”~ in a lysis buffer (RIPA). Lysats were immunopxxipitated using anti-FAK antibody. Samples were then run on SDS PAGE, transfered on nitroccllulose membrane, and FAK phosporylation was revealed by anti-tyrosine phosphorylated antibody. When tibroblasts were adherent cm the plastic at the tine of irradiation, FAK phosphmylation was detectable in sham irradiated cultures, and WA (2Jlcm~) stimulated this phosphorylation, confirming the rapid WA induced activation of intcgrins previously mentionned. Phosphorylation was less pronounced after lOJ/cmz of WA. When tihroblasts were cultured on a collaeen coatine. the WA-induced stimulation of FAK was mote marked even if the presence dthe mat&reduced the level of FAK phosphorylation m sham-irradiated tibmblasts. In addition, the presence of a collagen coating modified the intcgrins expression after WA irradiation. These results firstly show that the matrix modulates the WA-induced FAK phosphorylation, underlying a modulation of intcgrins activation. These results are to closcby to the fact that the extracellular matrix modify tibroblasts bchaviour, underlying the involvement of signal transduction depending on integrins in these effects.