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Abstracts of the Second Annual Meeting of the Canadian Society for Investigative Dermatology
ABSTRACTS
Abstracts ofthe Second Annual Meeting ofthe Canadian Society for Investigative Dermatology September 18, 1994. Chairman: Dr. Daniel N. Sauder AM 8:00 AM
Royal College Lectureship, "Molecular and Cellular Aspects ofthe Progression of Human Cutaneous Malignant Melanomas" Guest Speaker, Dr. R. Kerbel Abstract
General Plenary Session I 9:00 AM 9:15 AM 9:30 AM 9:45 AM 10:00 AM 10:15 AM
11:00 AM 11:15 AM
P53 Mutation in Metastatic Melanomas and Primary Melanomas from SunExposed and Sun-Protected Sites Detection of p53 Mutations in Benign and Dysplastic Nevi, and Metastatic Malignant Melanoma MIB-1 Proliferative Activity is a Significant Prognostic Factor in Primary Thick Cutaneous Malignant Melanomas Expression of Leukemia Inhibitory Factor (LIF) and Interleukin-11 (IL-11) by Human Melanoma Cell Lines: LIF, IL-6, IL-11 Are Not Coregulated Human Melanosome Specific Antigen (HMSA-6) Is Associated with the Expression of ER Network and Neural Crest Granules in Human Melanocytes and Melanoma Cells Predictable Changes in Differentiation of Human Melanoma Cells Induced by Bromodeoxyuridine and Melanocyte Stimulating Hormone Membrane "Differentiation" in Stratum Corneum Membrane Models Spectroscopic and Microscopic Properties of Skin Autofluorescence Emission
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PM General Plenary Session II 2:00 PM 2:15PM 2:30 PM 2:45 PM 3:15 PM 3:30 PM 3:45 PM 4:00 PM 4:15 PM
Skin Cytokine Profiles in Tight Skin (TSK) Mice with CD4 or CD8 Gene Disruption CD4 and CD8 Required for Optimal Development of Allergic Contact Dermatitis Inverse Dystrophic Epidermolysis Bullosa: Report of Two Cases with Further Correlation Between Immunofluorescence Studies and Electron Microscopy Localisation des Anticorps Anti-Membrane Basale dans la Pemphigoide Tumor Incidence and DNA Repair in UV Exposed p53 Transgenic Mice Tissue Distribution of UV-Inducible mRNA Transcripts in the Adult Mouse In Vitro Cytotoxicity of Phenolic Thioethers Biological Role of Tyrosinase-Related Protein-1 (TRP-1) in Melanogenesis in Co-Transfected Cells of Human Tyrosinase and TRP-1 Gene Manganese Induces Integrin-Mediated Human Melanoma Attachment and Spreading on Lower RGD Densities on Defined Substrates
0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc.
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131 P ^ Ml ITATION IN METASTATIC MELANOMAS AND PRIMARY MELANOMAS c D o u Ql N EXPOSED AND SUN-PROTECTED SITES. Victor A. Tron''. Gang L l ' ' . FROM s u ||^^,,r,,3n', Vlnnfipl C. Ho'. Division of Dermatology and Department of ^^t^^Z^TM^er^y o' British Columbia, Vancouver, Canada. raiiiuiuyj, ^^^ ^^^^ observed In many human malignancies, including skin cancers. P53 ' " " • a ^ ^^lg Ij, melanoma has been confllcllng. We have examined, by However ^^^^^^^ ^^l^g 1^^ PQ^ ^^ g j ^ , antibodies, the frequency of p53 overImmunonis ^^ metastatic meianomas and 61 primary meianomas. of which 30 are from expression ^^^^^ ^^ ^^ ^^^ 1^^^ sun-exposed sites. Ten of 14 metastatic melanomas 'h"''^lHn53 over-expression compared to oniy 8 of 61 primary meianomas (p<0.004). No snowea p ^^^^.g ^„ p53 expression was found between primary meianomas from sunsignitican ^^^ sun-exposed sites (6/31). We have aiso examined p53 mutation by protected i ^ / ^ ^.^nfirmation polymorphism (SSCP) anaiysis of 4 meianoma ceil lines. One sirig e ^ " ^ n^P^u^j jfoni a primary melanoma from a sun-protected site showed evidence ^fira^ltBred migration pattern in exon 7. Sequencing anaiysis of this region confirmed a ft Mtation in codon 244, showing a G to C transversion. This mutation is unlikely to point " ' " ' " " ^ radiation (UVR) since mutations caused by UVR are predominantly CC-TT r T transitions. In summary, p53 mutation in primary melanoma is uncommon and rt^ oear to be reiated to UVR. p53 mutation Is more common in metastatic does not ^PJ^^ ,|ng ,hat it may be involved as a late event In melanoma progression.
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132 DETECTION OF pS3 MUTATIONS IN BENIGN AND DYSPLASTIC NEVI, AND METASTATIC MALIGNANT MELANOMA. K S. Wilson. D. B. Levin. P. Kennv. G. V. de Amorim. J. Webber. British Columbia Cancer Agency. Victoria Clinic, University of British Coiumbia and Centre for Environmental Health. University of Victoria. Victoria, Point mutations in the tumour suppressor gene p53 are tiie most common genetic aiterations in human cancers, immunohistochemicai studies using frozen and/or formalinfixed sections of benign nevi, primary melanoma, and metastatic melanoma, as well as a number of melanoma celi iines, have yielded confiicting results on the stage at which altered P53 proteins appear in the development of melanoma and the frequency of aitered proteins in each stage. We have examined premaiignant and maiignant meianocytic ceiis derived directiy from fresh biopsy tissue for the presence cf p53 mutations. Using seiective media for melanccyte growth, short term primary cuitures enriched for melanocytes from skin biopsies of common acquired (benign) nevi, dyspiastic nevi, and metastatic meianoma were estabiished. Using the poiymerase chain reaction and single-stranded conformationai poiymorphism analysis, we detected p53 mutations in 3 of 11 benign nevi, 2 of 5 dyspiastic nevi, and 2 of 8 metastatic meianoma sampies. Of the 3 benign nevi that were positive for p53 mutation, one was derived from a patient with previous maiignant meianoma and another from a patient with a famiiy history of meianoma, both established risk factors for malignant melanoma. Two different p53 mutations were identified in 2 biopsies from a patient with metastatic melanoma. These results suggest that p53 mutation may be a very eariy event in the development of melanoma.
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MIB 1 PROLIFERATIVE ACTIVITY iS A SiGNIFICANT PROGNOSTIC FACTOR IN PRIfWARY THICK CUTANEOUS MALIGNANT MELANOMAS, L From, JA Ramsay, HJ K hn NA Iscoe Departments of Medicine & Pathology, University of Toronto, Women's Colieoe Hospital & Toronto-Bayview Regionai Cancer Centre, Toronto, ON, Canada The biological behaviour of primary cutaneous melanoma is extremeiy variable and whiie the Breslow verticai thickness measurement is the most significant factor in determining oqnosis there are stili thin meianomas that metastasize and thick meianomas which do not Another possible significant prognostic factor is the mitotic rate, A more sensitive rf-iorminafion of proliferation is the immunohistochemical demonstration of the Ki-67 nuc^ea an igen which is present in proliferating cells throughout the cell cycle (G,, S, G,, Ml MIB-1 a new murine monoclonal antibody to an epitope of the Ki-67 antigen, can be used on formalin-fixed paraffin-embedded archival tissue uniike Ki-67 antibody which can only be detected on frozen tissue, Althouqh the survival rate in thick melanomas is generally poor, it is nevertheless unpredictable Proliferation rate may be a significant biologic marker in this group, Clinicai staae I meianoma patients with tumours a 4 mm thickness were identified in our large melanoma database In the 64 tumours obtained for study, proiiferation was assessed by mitotic index and percentage of MIB-1 positivity and was then correiated with survivai. The median follow-up time was 3.8 years (range 0.4 to 13,6 years). The percentage of MIB 1 positivity was < 10% in 33 cases, 10-20% in 17 cases, and > 20% in 14 cases. Melanomas with the highest MiB-1 positivity (ieveis > 20%) were associated with a siqnificantiy poorer cause-specific survivai as compared to those patients who had tumours wKh intermediate (p ^ 0,0001) or iow levels of MIB-1 positivity (p = 0,0025). Muitivariate analysis using the Cox regression modei demonstrated that MIB-1 positivity was a significant independent prognostic factor reiated to cause-specific survival (p = 0,0002) and was more sensitive than tumour thickness or mitotic index in this seiect group of high nsk patients. Further studies are necessary to evaluate whether this finding is equaily significant in all melanomas.
EXPRESSION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND INTERLEUKIN11 (IL-11) BY HUMAN MELANOMA CELL LINES: LIF, IL-6, IL-11 ARE NOT COREGULATED. D Paelia. T. Kono. F, Bennett'. K, Turner'. C, Lu'. R S , Kerbel'. D N . Sauder. R C McKenzie. Divisions of Dermatology and 'Cancer Research, Sunnybrook HSC, University of Toronto, Toronto, and 'Genetics Institute, Cambridge, MA. Autocrine cytokine production is thought to be important in progression of neoplastic melanocytes towards metastatic malignancy. Melanoma lines established from human radial or vertical growth phase tumors are growth-inhibited by IL-6, as well as by Oncostatin M. In metastatic lines the sensitivity to both cytokines is lost (Cancer Res,53:2708,1993). We hypothesized that in malignant melanomas there may be coregulation of response to lL-6 and the other cytokines which use the same gpl30 signal transduction system, namely, LIF and IL-11. Early-stage to advanced-stage melanoma lines (the WM8 series) were incubated with 0.001-100 ng/mL LIF and IL-11. No proliferative or inhibitory response to these cytokines was observed by ('H)-thymidine incorporation. LIF and IL-11 mRNA and protein expression was measured by reversetranscription polymerase chain reaction (RT-PCR) and bioassay from early-stage and advanced-stage melanoma lines. Whereas all the lines except one (MeWo) produced LIF mRNA and protein, only two lines (WM 793 and WM 983A) produced signiflcant IL-11 mRNA and protein despite the presence of smalt, but detectable amounts of IL-11 message in all the other lines. The lines secreting highest levels of LIF (WM 983), IL6 (MeWo) and IL-11 (WM 793) were different. No evidence could be found for either co-regulation of expression of these cytokines or the cellular responses of each individual line to them. However, there appears to be higher rates of mRNA and protein expression of LIF, IL-11 and IL-6 in the advanced-stage melanomas.
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PREDICTABLE CHANGES \N DIFFERENTIATION OF HUMAN MELANOMA CELLS HUMAN MELANOSOME SPECIFIC ANTIGEN (HMSA-6) IS ASSOCIATED WTffl THE INDUCED BY BROMODEOXYURIDINE AND MELANOCYTE STIMULATING HORMONE. EXPTJESSICW OF ER N E W D R K AND NEURAL CREST C3RANULES IN HUMAN MELANOCYTES GE Searles. H Chen. K Jimbow. Div.Derm.Cutan.Sci,U.of Albena, Edmonton, AB, AND MELANCMA CEIiS, H, Ctien. K, Jimbow. Div, of Dermatology & Cut:aneous Canada Sciences, University of Alberta, Eamonton, CANATA, The differentiation state of malionant celts correlates with metastatic potential in In order to characterize melanogenesis-associated proteins, we have many cell lines. Halogenated pyrimidine analogues like bromodeoxYuridine (BRDU); and recently developed several monoclonal antibodies, MoAbs, using melanoscmes growth hormones like a-Metanocyte Stimulating Hormone (a-MSH) can reversibly isolated from human melanocytes and melanoma cells. One of the recently established antibodies, MoAb HMSA-6 was found to be unique because it has change differentiation. We determined the effect of differentiation induction by these 4 antigenic epitopes with sizes of 11, 17, 22 and 41 kDa, that these 4 can agents on changes in morphology (cell size, shape, dendricity, melanosome number), cell proliferation, soft agar colony formation, and pigmentation on 4 non-metastatic be grouped into 2 species, (a) 41 kDa, and (b) 11, 17 and 22 kDa, after human malignant meianoma (HMM) cell lines, (WM35, WM39, WM902B, WM1341D) V8 protease digestion. This study has conducted the immunohistochemical and 4 metastatic HMM cell lines (SK-MEL23, SK-MEL24, G361, C32). ctiaiacteristics of MoAb HMSA-6 epitope distribution, using FITC labelled After 7 days in 1 8^M BRDU, all HMM, except WM1341D, were less differentiated indirect immunofluorescence and laser scan confocal microscc^jy, TVo patterns of immunofluorescence were seen in both normalroelanocytesand (25-99% less tyrosinase activity, reduced TRP-1 content, and melanin; 3-5x higher colony formation). However, WM1341D was more differentiated (3x more tyrosinase melaiKima cells, including amelanotic cells, Ihey were (a) reticular or activity and melanin; 3x fewer colony formations). No cell shape changes were filamentous network located in both perinuclear areas and dendrites, and observed, except WM1341D, which became 5x larger and highly dendritic. (b) round or granular perinuclear aggregates. Basically, similar findings After 7 days in 1O'^M a-MSH, all HMM, except WM35, were unchanged in all were obtained in human neuroblastoroa cells. In contrast, human fibroblasts revealed prinarily the fibrillar or reticular networks extending to the parameters compared to control. WM35 became more differentiated than control (lOx more tyrosinase activity, and melanin content; more dendritic). perikaryon and dendrite, T^e control specimen stained with antibodies against 10 nm vimentin filament and tubulin revealed entirely different We show that:1) BRDU and a-MSH can variably alter differentiation in all HMM; 2) patterns of filamentous network, indicating that HMSA-6 is not related to, WM1341 D becomes more differentiated with BRDU induction, while all other cell tines or associated with 10 nm vimentin or microtubules. We propose tiiat become less differentiated; 3) WM35 can become either more or less differentiated by filamentous or reticular pattem of HMSA-6 immunostaining is associated using a-MSH, or BRDU, respectively. WM35 and WM1341D could be very useful with the networks of endoplasmic reticulum and its granular perinuclear models for examining gene expression during differentiation in melanoma cell tines, deposits are related to neurosecretory granules common to both melanocytic especially WM35, since 1) it was derived from a radial-growth phase tumour, and 2) is cells and neuroblastoma cells. the most plastic of all cell tines tested with regard to differentiation.
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MEMBRANE "DIFFERENTIATION" IN STRATUM CORNEUM MEMBRANE MODELS. Neil KHson and Jenifer Thewalt, Division of Dermalology and Department of Physics , University of British Columbia. During epidermal differentiation, monoglucosylceramides are converted to ceramides by B-glycosidases. This lipid modification is one of a number that contributes to the formation of the stratum corneum interceilular membranes which are in turn responsible for the epidermal permeability barrier. We have previously shown by deuterium magnetic resonance and X-ray diffraction that modei membranes of composition simiiar to that found in the SC {i.e.. ceramide:chciesterol:palmitic acid 1:1:1 mol/moi) are composed of large crystalline domains co-existing with smaller fiuid domains at physiological temperatures, and we speculated that such architecture resulted In a porous medium through which diffusion may be described by percolation. In certain tissues (e.g., buccal mucosa) and disorders (Gaucher's Disease) the activity of (i-gtycosidases Is reduced or absent, and this is accompanied by a rate of transepithelial water loss that is greater than that observed in normal skin. We therefore examined model membranes composed of bovine brain monoglucosylceramide, cholesteroi, and perdeuterated palmitic acid (1:1:1 mol/mol) at pH 5.2. We found that solid domains were formed at room temperature, but that these were smaller than those found with ceramide, and transition to a fluid lamellar signals occurred at correspondingly lower temperatures. We believe that these results are consistent with the hypothesis that tissue properties, in this case transepidermai water loss, can be related directly to lipid paci
SPECTROSCOPIC AND MICROSCOPIC PROPI-RTIES Or SKIN AUTOFLUORl-SCENCE EMISSION. HaJRhan Zcnt;. Cnliim MacAulav. David 1. McLcnii*. llarvtfV Lui*. Orflnko Palcic. Cancer Imaging Department and *Division of Dcrmatoiogic Oncology, British Columbia Cancer Agency, Vancouver. To improve the understanding of human skin autofluoresccnce emission, the spectroscopic und microscopic charaeteristies of skin aulofluorcscencc were studied using a combined fluorescence and reflectance spectroanalyser and a fiber optic microspectrophotomcter. The aulofluoreseence spectra of in vivo human skin were measured over a wide range of" excitation wavelengths (350 nm - 470 nm). It was found that not only the intensity but also the sliape of these speetra were exeitation wavelength dependent. The excitation emission matrices (EEMs) of in vivo skin were constructed from these data. An exeitation emission maximum pair (380 nm, 470 nm) was identified. The most probable energy of skin autofluorescenee emission photons tends to increase linearly with increasing exeitation photon energy. We also demonstrated that the diffuse refieetanee (R) ean be used as a first order approximation of the fluoreseenee distortion faetor (/) to correct the measured in vivo autofluorescenee spectra for the effeet of tissue reabsorption and scattering. The microscopic in vitro autoOuorescenee properties of excised skin tissue sections were examined using 442 nm 1 ie-Cd laser light exeitation as an example. It was revealed that the fluorophore distribution inside the skin tissue is not uniform. The autofluorescenee speetra of different anatomical skin layers are also different. This study suggested that the major skin ttuorophores are located in the dermis. However, the exact fluorophore moiecuies have yet to be identified. Nevertheless, these mieroscopic data allowed us to reconstruct the 442 nm laser light exeited in vivo (macroscopic) autofluorescenee speetrum of skin tissue using Monte Carlo simulation.
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SKIN CYTOKINE PROFILES IN TIGHT SKIN (TSK) MICE WITH CD4 OR CD8 GENE DISRUPTION. Daniel N. Sauder. Takeshi Kono. Seiii Kondo. Savetia Pastore. Valerie A. Wallace. Christopher J. Paipe. Tak W. Mak and Roderick C. McKenzie. Division of Dermatology, Department of Immunology and Biophysics, University of Toronto, Toronto, Canada.
CD4 AND CD8 REQUIRED FOR OPTIMAL DEVELOPMENT OF ALLERGIC CONTACT DERMATITIS. Seiii Kondo. Gulnar M. Shivii. Takeshi Kono. Saveria Pastore. Tak W. Mak. and Daniel N. Sauder. Division of Dermatology and Department of Biophysics, University of Toronto, Toronto, Canada The use of gene targeting has provided powerful tool to examine precise functional roles of molecules in the immune system. In order to study the role of CD4 and CD8 molecules in the development of allergic contact dermatitis (ACD), the mutant mice lacking CD4 or CD8 were generated using gene targeted disruption. The mutant mice and syngeneie controls (C57BL/6) were sensitized on the abdominal skin with DNFB and challenged on the ears 6 days later. Ear swelling was assessed as a measure of ACD. Mice lacking CD4(-) had a marked hyporesponsiveness of 38.3±9.0% (P<0.025) in ear increment when compared with normal control. CD8(-) mice also showed hyporesponsiveness (67.8±7.0%, P<0.025) but to a lesser extent than CD4(-) mice. Skin.cytokine profiles after challenge were analyzed in CD4(-) mice by RT-PCR. Total RNA was extracted from the ear at various time points after hapten application to sensitized mice, reverse transcribed to cDNA, and amplified by PCR using labeled specific primers. Cytokine signal densities were expressed relative to p-actin signals and these ratios were compared to C57BL/6 controls. In normal mice, IFN-y, IL-6 and IL-10 mRNA were upregulated between 6-24 h after hapten application (42, 22 and 23fold, respectively at 24 h), while in CD4(-) mice these genes were not upregulated to the same extent (17, 17 and 15-fold, respectively). These results suggest that both CD4 and CD8 molecules are required for optimal induction of ACD. The CD4 molecule may influence the development of ACD by modulating the cytokine profiles in the skin.
The tight skin (Tsk) mice (dominant mutation in C57B1/6) represent a murine model of heritable fibrosis with some similarities to human scleroderma. To determine the role of T-cell subtypes in cutaneous fibrosis, the Tsk mice lacking CD4 or CD8 were generated using gene-targeting disruption. We have already shown that Tsk/CD4 (-) mice show less severe skin fibrosis compared with CD4(+)/CD8(+) or CD8(-) Tsk mice. In this study, we analyzed skin cytokine profiles by RT-PCR. Total RNA was extracted from Tsk, CD4 or CD8 gene-targeted Tsk and normal C57B1/6 mice, reverse transcribed to cDNA, and amplified by PCR using labelled specific primers. Cytokine signal densities were expressed relative to p-actin signals, and these ratios were compared to C57B1/6 controls (C57B1/6 ratio = 1 ) . In Tsk mice with severe fibrosis, TGF-P, bFGF and PDGF-A mRNA were upregulated (2.6 ± 0.4; 1.8 + 0.2 and 2.7 ± 0.2, respectively; P<0.05), whereas Tsk/CD4(-) mice with mild fibrosis had normal level of TGF-P and PDGF-A but suppressed level of IL-la, TNF-a and bFGF (0.5 + 0.1; 0.3 + 0.1 and 0.2 + 0.1, respectively; P<0.05). Tsk/CD8(-) mice had severe fibrosis but had normal levels of IL-la, bFGF and PDGF-A but TGF-P mRNA was elevated ((2.1 + 0.7, P<0.05). These results suggest that alteration in fibrogenic cytokines, especially TGF-P, is correlated with fibrosis in Tsk mice and that CD4 gene may influence fibrotic change in Tsk.
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INVERSE KfSTRDPHIC EPIDERMOLYSIS BULLOSA! REPORT OF 1W0 C3\SES WITH FURTHER OQEBEtATION BBIWEEN IMMUNOFLUORESCENCE STUDIES 2^ND ELECTRON MICBDSOOPX. A. Lin*. L.T. Smith**. J.D. Fine***. *The Rockefeller University, New York, NY. **University of Washington School of Medicine, Seattle, WA. ***University of North Carolina at Chapel Hill, NC. We present two patients with dystirophic EB whose lesions occurred in stir iking inverse distribution. A 17 year-old boy presented 4 years ago with blisters mainly in the groin, ankyloglossia, dysphagia and microstoroia which subsequently required plastic surgical reconst:ruction. Barium swallow showed proximal esophageal narrowing. Biopsy of a fresh blister showed separation below the lamina densa and diminished anchoring fibrils Oh electron microscopy, but n o m a l staining of LH 7:2 on immunofluorescence. A 9 year-old boy presented 4 years ago with blisters mainly in the axillae and groin, short frenulum and no dysphagia. Electron microscopy had previously shown separation below the lamina densa, and repeat biopsy showed sparse immature anchoring fibrils but normal staining with IH 7:2 antigen. These findings suggest that inverse dystrophie EB is a rare, unique subset characterized by normal M 7:2 staining but diminished anchoring fibrils, variable clinical course, and marked oral and esophageal involveinent in some patientis. It may possibly be caused by a point mut:ation in tiie type VII collagen gene at a site whose ej^tession is not detected by the monoclonal antibody LH 7:2.
LOCALISATION DES ANTICORPS ANTI-MEMBRANE BASALE DANS LA PEMPHIGOIDE. B. Mavnard. Service de Dermatoloyie, Di5partement de M^ecine, Centre Hospitaller Universitaire, UniversitiS de Sherbrooke, Sherbrooke. Leu maladies du groupe de la pemphigoide et celles du groupe de I'^pidermolyse bulleuse acquise (EBA) peuvent se traduire par un tableau clinique et histopathologique semblable et par une immunopathologie de routine identique. La technique du clivage de la membrane basale par le NaCl permet de s6parer physiquement les antig&nes de la pemphigoide (composants des hdmidesmosomes, retrouvds du cfit^ ^pidermique de la separation) de I'antigfene de I'EBA (le collag&ine de type VII constituant des fibrilles d'ancrage, retrouv^ du c6t^ dermique de la siSparation). L'immunofluorescence indirecte sur ce substrat a permis de caractdriser les anticorps circulunts contenus dans les scrums de 7 patients avec pemphigoide bulleuse et 2 patients avec pemphigoide cicatricielte. Tous les scrums testes (100%) ont montr6 un patron ^pidermique (toit de la buUe). Le titre des unticorps circulants fut determine sur oesophage de singe et sur peau humaine dissociee dans 6 cas. Dans tous les cas le titre titait plus 6leviS sur In peau dissociiSe, La technique de I'^piderme dissoci^ est simple et permet de caracttSriser les anticorps circulants dans la pemphigoide. La prevalence de I'EBA est faible dans notre milieu. La peau dissoci^e est un substrat plus sensible que l'oesophage de singe pour diStecter des anticorps circulants.
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VOL. 103, NO. 2 AUGUST 1994
143 T TUMOR INCIDENCE AND DNA REPAIR IN UV EXPOSED P53 TRANSGENIC MICE G Li''. D Mitchell. VC H(i- and VA Tron'-. Du partment of TRANSGENIC Division MICE. ol Dermatology^, University of British Columbia, BC und M,D. iii P tholoijv' Anderson Cancer Center'' University of Texas, Texas. n53 is an important tumor -suppressor, p53 protein is thought to regulate the cell cycle after a DNA damage event. Mutations ot p53 geno have been implicated in the oathocenesis of cutaneous squamous cell carcinoma (SCC), To examine the role of p53 in skin carcinogenesis, we observed the development of skin cancers in p53 transjjenic mice, which overexpress mutant alleies of the p53 gene, and control mice with or without UVB for 34 weeks. 28 tumors developed in 19 p53-UV irradiated mice versus 14 of 18 in the control vroup- 9 p53 niice but none of the control mice developed multiple tumors. Histologically, most tumors were spindle cell malignancies, consistent with spindle cell squamous cell carcinomas. The mean time to appearance of a tumor (minimum of 5mm in diameter and histologically proven malignancy), did not difter between the two groups (p = 0.16). To determine whether the presence of p53 mutations alters DNA repair, cyclobutane diniers and 6-4 photoproducts were assayed in skin after exposure to UV radiation and expressed as % remaining photoproduct post radiation. The p53 transgenic animals demonstrated decreased repairof cyclobutane dimers at 4 hrs (control 55±9 vs. p53 103±6, p = 0,0002*) and 24 hrs fcontrol 56 + 11 vs. p53 77ji7, p = O.OOI *), and reduced repair of 6-4 photoproducts at 4 hrs (control 3 5 ^ 8 vs. p53 85±7, p = 0.0001*). Our findings indicate that mutant p53 predisposes mice to multiple and increiised numbers of UV-induced tumors, but does not shorten the latency period for tumor development. Multiple and increased numbers of tumors in p53 transgenic mice may be the result of reduced DNA repair. *(meanJiSEM)
145 TRO CYTOTOXICiTY OF PHENOLIC THIOETHERS. A.F-. Gili, M, Tiincion, T, ~K. Jinihow. University of Alberta. Division of Dermatology and Cutaneous Sciences. Edmonton. Alberta, We have recently synthesized two new phenolic thiocthcrs in our laboratory, N-propii)nyl-4-Scysteaminyl phenol (NPr-4S-CAP) ;ind N-{2-|(4-propionyloxypheny!)thio]etliyl}propioiunnidc fNPP-TEP) These compounds are tyrosine analojjucs, similar to other previously deseribed phenolic thiocthers such as 4S-cysteaminyl phenol (4S-CAP) ;ind N-acetyl-4S-cyte:iminyl phenol (NAC-4S-CAP). The cylotoxieity of these latter two compounds have been proposed to be primarily tyrosinasc dependent, whieh may make them suitable as specific anti-metanonia therapy sinee only melanocytes and their malignant counterparts ctintain this active enzyme. To further investigate this hypothesis, we preformed MTf (3-(4.5-diiTieihylthiazol-2-yl)-2.5diphenyltetrazolium bromide) assays as a method of determining cytotoxiciiy on 9 different cell lines using the NPr-4S-CAP and NPP-TEP compounds. We also performed the same assays using NAe-4S-CAP and a pro-drug, N-{2-[(4-acetoxyphenyl)thiojethyl}acetaniide (NAP-TEA), which was also developed in our laboratory. The cells were exposed to the drug for two days. Of the nine cell lines, all the human neuroblastoma lines (SK-NMC. SK-NSH, KAN) and an amelanotic human melanoma line (C32) were consistently most sensitive to all the drugs. The consistently most resistant cell lines included SK-MBL-23 (highly pigmented human melanoma), SK-MEL-24 (amelanotic human melanoma) and human fibroblasts. The eell lines in the middle range of sensitivity were HeLa (human cer\'ieal eanccr) and G.Vji (human melanoma with low tyrosinase activity). Our present in viiro study may indicate ihat there is a non-tyrositiase mediated eytotoxicity for phenolie thioethers in addition to the lyrosinase mediaicd one as previously proposed.
147 MANGANESE INDUCES INTEGRIN-MEDIATED HUMAN MELANOMA ATTACHMENT AND SPREADING ON LOWER RGD DENSITIES ON DEFINED SUBSTRATES. GE Searles. K Jimbow.Div.Derm.Cutan.Sci,U. of ALberta, Edmonton, AB, Canada Glv-Arg-Gly-Asp-Ser (GRGDS) is a ligand for several integrins expressed on human malignant melanocytes, including tha metastatic malignant melanoma cell line, G361. To determine the effect of cations on the minimal number of ligand-receptor interactions required for G361 adhesion, GRGDS was covalently grafted at densities ranging from 0,1 to 10* fmol/cm^ to poorly adhesive glass substrates, Cell morphologies were determined in serum-free media (SFM) with or without manganese (Mn'^1 present. Perturbation of adhesion by functional anti-integrin antibodies was determined, as was the sensitivity of adhesion to several cytoskeletal inhibitors. G361 adhesion to GRGDS was divalent cation-dependent, and increased with increasing GRGDS densities. Adhesion was greater with Mn"^ (1 mMl compared to SFM at each GRGDS density. Optimal cell spreading occurred at an RGD density of 10 fmol/cm' with SFM, and at 1 fmol/cm* with Mn"^ present. With SFM, adhesion was inhibited with anti-a3 (76%) and anti-Bl (97%) antibodies; with M n ' ^ anti-a2 (34%), anti-CT3 (90%), anti-o5 (15%) and anti-GI (93%) antibodies were inhibitory. Increased membrane viscosity (paraformaldehyde) had equivalent inhibition (70%) in both cation conditions; whereas actin depolymerization (cytochalasin D), Integrin-cytoskeletal unlinkage (dibucalne) and glycolysis inhibition (sodium fluoride) was more inhibitory for G361 cells with Mn'^ present, than with SFM alone. We show that; 1) adhesive peptides can be covalently grafted in a quantitative manner; 2) G361 cells can bind to this substrate in a divalent cation-dependent fashion; 31 manganese permits adhesion at lower RGD densities, probably through recruitment of o2B1 and o5fi1 integrins and increased cytoskeletal organization. This fully-defined substrate will be useful In precise quantification of each component of the adhesion cascade.
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144 TISSUE DISTRIBUTION OF UV-INDUCIBLE mRNA TRANSCRIPTS IN THE ADULT MOUSE C. Rosen. R. Poon. R. Rathee. D. Drueker. Department of Medicine, Women's College Hospital and Toronto General Hospital, University of Toronto, Toronto. We have recently reported the isolation of 23 keratinocyte mRNA transcripts that were induced by ultraviolet B radiation in neonatai rat keratinocytes in vitro. To determine whether the novel UVB-induced genes were also expressed in non-cutaneous tissues in vivo, we have isolated RNA from a variety of Skh-1 hairless mouse tissues, and characterized the tissue distribution of gene expression for 15 of the 23 genes in wVo using Northern blot analysis. Several distinct patterns of gene expression were identified. Ali of the genes were expressed in mouse skin. Two genes were strongly expressed in skin with only minimal expression detected in other tissues. Four genes were expressed in all tissues studied, pancreas, liver, spleen, kidney, small and large bowel, lung, heart and skin. Two genes were expressed oniy in the skin and the gastointestinai tract. Generally, the pancreas showed the lowest level of transcript expression and four genes were expressed in ali tissues except the pancreas.These observations demonstrate that genes originally isolated from a keratinocyte cDNA library are generally expressed in a diverse number of mouse tissues, suggesting that the proteins encoded by the majority of these UVB-induoed cDNAs likely subserve distinct ceiiuiar functions not restricted to the biology of the keratinocyte In vivo.
146 BIOUOGIGRL ROLE OF TYPOSINASE-REIATED PROTElN-1 (TRP-1) DJ MEIANDGENESIS' IN CtHTRANSFECTED CELLS OF HUMAN TYRDSINASE AND TRP-1 GENE. K. Jimbow, D. Ijuo. H. Chen, Div. of Deniiatolagy & Cutaneous Sciences, University of Alberta, Edmonton, CANADA, Tyrosinase is a key enzyme responsible for melanogenesis. Recently however, 2 tyrosinase-related proteins (TRP-1, 2) and encoding genes have been identified. Tt^P-2 has been attributed to dopachrome tautomerase which converts dopachrome to dihyrdoxindole 2 carboxylic acid to fortn eumelanin. In contrast, the nature of TRP-1 is still unclarified. In order to characterize the possible biological role of TRP-1 in melanogenesis, we constructed 2 genes specific to melanogenesis, hunan tyrosinase (HT) and TRP-1, into monkey kidney oos-7 cells and human amelanotic melanoma cells. Transfectants were examined for mRNA expression by reverse transcription mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectant or co-transfectant. Both light and electron microscope observation indicated that degeneration and premature death of melanocytes occurred by HT transfection, but not by TRP-1 alone or by combination of both. Co-transfected cells from amelanotic melanoma cells and COS-7 cells revealed a granular product which reacted with a specific anti-TRP-1 antibody and exhibited numerous lysosomal vacuoles with electron-dense material, indicating aggregation/degradation of new protein product, as well as melanin in lysosomal vacuoles. Our melanin assay confirmed new production of melanin in these cells. There was also a dranatic elevation of gene and protein expressions of lanp-l (lysosomeassociated membrane protein-1) in co-transfected cells. TRP-1 gene may, therefore, f unct ion together with Lairp-l by stabi 1 iz ing the enzymic conplex of tyrosinase in melanogenesis in order to have proper sequence of melanogenesis events as well as preventing tyrosinase-mediated cytotoxicity.
Abstracts ofthe Second Annual Meeting ofthe Canadian Society for Investigative Dermatology September 18, 1994. Chairman: Dr. Daniel N. Sauder AM 8:00 AM
Royal College Lectureship, "Molecular and Cellular Aspects ofthe Progression of Human Cutaneous Malignant Melanomas" Guest Speaker, Dr. R. Kerbel Abstract
General Plenary Session I 9:00 AM 9:15 AM 9:30 AM 9:45 AM 10:00 AM 10:15 AM
11:00 AM 11:15 AM
P53 Mutation in Metastatic Melanomas and Primary Melanomas from SunExposed and Sun-Protected Sites Detection of p53 Mutations in Benign and Dysplastic Nevi, and Metastatic Malignant Melanoma MIB-1 Proliferative Activity is a Significant Prognostic Factor in Primary Thick Cutaneous Malignant Melanomas Expression of Leukemia Inhibitory Factor (LIF) and Interleukin-11 (IL-11) by Human Melanoma Cell Lines: LIF, IL-6, IL-11 Are Not Coregulated Human Melanosome Specific Antigen (HMSA-6) Is Associated with the Expression of ER Network and Neural Crest Granules in Human Melanocytes and Melanoma Cells Predictable Changes in Differentiation of Human Melanoma Cells Induced by Bromodeoxyuridine and Melanocyte Stimulating Hormone Membrane "Differentiation" in Stratum Corneum Membrane Models Spectroscopic and Microscopic Properties of Skin Autofluorescence Emission
131 1S2 ' 133 : 134 ;: 135 136 137 138
PM General Plenary Session II 2:00 PM 2:15PM 2:30 PM 2:45 PM 3:15 PM 3:30 PM 3:45 PM 4:00 PM 4:15 PM
Skin Cytokine Profiles in Tight Skin (TSK) Mice with CD4 or CD8 Gene Disruption CD4 and CD8 Required for Optimal Development of Allergic Contact Dermatitis Inverse Dystrophic Epidermolysis Bullosa: Report of Two Cases with Further Correlation Between Immunofluorescence Studies and Electron Microscopy Localisation des Anticorps Anti-Membrane Basale dans la Pemphigoide Tumor Incidence and DNA Repair in UV Exposed p53 Transgenic Mice Tissue Distribution of UV-Inducible mRNA Transcripts in the Adult Mouse In Vitro Cytotoxicity of Phenolic Thioethers Biological Role of Tyrosinase-Related Protein-1 (TRP-1) in Melanogenesis in Co-Transfected Cells of Human Tyrosinase and TRP-1 Gene Manganese Induces Integrin-Mediated Human Melanoma Attachment and Spreading on Lower RGD Densities on Defined Substrates
0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc.
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131 P ^ Ml ITATION IN METASTATIC MELANOMAS AND PRIMARY MELANOMAS c D o u Ql N EXPOSED AND SUN-PROTECTED SITES. Victor A. Tron''. Gang L l ' ' . FROM s u ||^^,,r,,3n', Vlnnfipl C. Ho'. Division of Dermatology and Department of ^^t^^Z^TM^er^y o' British Columbia, Vancouver, Canada. raiiiuiuyj, ^^^ ^^^^ observed In many human malignancies, including skin cancers. P53 ' " " • a ^ ^^lg Ij, melanoma has been confllcllng. We have examined, by However ^^^^^^^ ^^l^g 1^^ PQ^ ^^ g j ^ , antibodies, the frequency of p53 overImmunonis ^^ metastatic meianomas and 61 primary meianomas. of which 30 are from expression ^^^^^ ^^ ^^ ^^^ 1^^^ sun-exposed sites. Ten of 14 metastatic melanomas 'h"''^lHn53 over-expression compared to oniy 8 of 61 primary meianomas (p<0.004). No snowea p ^^^^.g ^„ p53 expression was found between primary meianomas from sunsignitican ^^^ sun-exposed sites (6/31). We have aiso examined p53 mutation by protected i ^ / ^ ^.^nfirmation polymorphism (SSCP) anaiysis of 4 meianoma ceil lines. One sirig e ^ " ^ n^P^u^j jfoni a primary melanoma from a sun-protected site showed evidence ^fira^ltBred migration pattern in exon 7. Sequencing anaiysis of this region confirmed a ft Mtation in codon 244, showing a G to C transversion. This mutation is unlikely to point " ' " ' " " ^ radiation (UVR) since mutations caused by UVR are predominantly CC-TT r T transitions. In summary, p53 mutation in primary melanoma is uncommon and rt^ oear to be reiated to UVR. p53 mutation Is more common in metastatic does not ^PJ^^ ,|ng ,hat it may be involved as a late event In melanoma progression.
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132 DETECTION OF pS3 MUTATIONS IN BENIGN AND DYSPLASTIC NEVI, AND METASTATIC MALIGNANT MELANOMA. K S. Wilson. D. B. Levin. P. Kennv. G. V. de Amorim. J. Webber. British Columbia Cancer Agency. Victoria Clinic, University of British Coiumbia and Centre for Environmental Health. University of Victoria. Victoria, Point mutations in the tumour suppressor gene p53 are tiie most common genetic aiterations in human cancers, immunohistochemicai studies using frozen and/or formalinfixed sections of benign nevi, primary melanoma, and metastatic melanoma, as well as a number of melanoma celi iines, have yielded confiicting results on the stage at which altered P53 proteins appear in the development of melanoma and the frequency of aitered proteins in each stage. We have examined premaiignant and maiignant meianocytic ceiis derived directiy from fresh biopsy tissue for the presence cf p53 mutations. Using seiective media for melanccyte growth, short term primary cuitures enriched for melanocytes from skin biopsies of common acquired (benign) nevi, dyspiastic nevi, and metastatic meianoma were estabiished. Using the poiymerase chain reaction and single-stranded conformationai poiymorphism analysis, we detected p53 mutations in 3 of 11 benign nevi, 2 of 5 dyspiastic nevi, and 2 of 8 metastatic meianoma sampies. Of the 3 benign nevi that were positive for p53 mutation, one was derived from a patient with previous maiignant meianoma and another from a patient with a famiiy history of meianoma, both established risk factors for malignant melanoma. Two different p53 mutations were identified in 2 biopsies from a patient with metastatic melanoma. These results suggest that p53 mutation may be a very eariy event in the development of melanoma.
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MIB 1 PROLIFERATIVE ACTIVITY iS A SiGNIFICANT PROGNOSTIC FACTOR IN PRIfWARY THICK CUTANEOUS MALIGNANT MELANOMAS, L From, JA Ramsay, HJ K hn NA Iscoe Departments of Medicine & Pathology, University of Toronto, Women's Colieoe Hospital & Toronto-Bayview Regionai Cancer Centre, Toronto, ON, Canada The biological behaviour of primary cutaneous melanoma is extremeiy variable and whiie the Breslow verticai thickness measurement is the most significant factor in determining oqnosis there are stili thin meianomas that metastasize and thick meianomas which do not Another possible significant prognostic factor is the mitotic rate, A more sensitive rf-iorminafion of proliferation is the immunohistochemical demonstration of the Ki-67 nuc^ea an igen which is present in proliferating cells throughout the cell cycle (G,, S, G,, Ml MIB-1 a new murine monoclonal antibody to an epitope of the Ki-67 antigen, can be used on formalin-fixed paraffin-embedded archival tissue uniike Ki-67 antibody which can only be detected on frozen tissue, Althouqh the survival rate in thick melanomas is generally poor, it is nevertheless unpredictable Proliferation rate may be a significant biologic marker in this group, Clinicai staae I meianoma patients with tumours a 4 mm thickness were identified in our large melanoma database In the 64 tumours obtained for study, proiiferation was assessed by mitotic index and percentage of MIB-1 positivity and was then correiated with survivai. The median follow-up time was 3.8 years (range 0.4 to 13,6 years). The percentage of MIB 1 positivity was < 10% in 33 cases, 10-20% in 17 cases, and > 20% in 14 cases. Melanomas with the highest MiB-1 positivity (ieveis > 20%) were associated with a siqnificantiy poorer cause-specific survivai as compared to those patients who had tumours wKh intermediate (p ^ 0,0001) or iow levels of MIB-1 positivity (p = 0,0025). Muitivariate analysis using the Cox regression modei demonstrated that MIB-1 positivity was a significant independent prognostic factor reiated to cause-specific survival (p = 0,0002) and was more sensitive than tumour thickness or mitotic index in this seiect group of high nsk patients. Further studies are necessary to evaluate whether this finding is equaily significant in all melanomas.
EXPRESSION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND INTERLEUKIN11 (IL-11) BY HUMAN MELANOMA CELL LINES: LIF, IL-6, IL-11 ARE NOT COREGULATED. D Paelia. T. Kono. F, Bennett'. K, Turner'. C, Lu'. R S , Kerbel'. D N . Sauder. R C McKenzie. Divisions of Dermatology and 'Cancer Research, Sunnybrook HSC, University of Toronto, Toronto, and 'Genetics Institute, Cambridge, MA. Autocrine cytokine production is thought to be important in progression of neoplastic melanocytes towards metastatic malignancy. Melanoma lines established from human radial or vertical growth phase tumors are growth-inhibited by IL-6, as well as by Oncostatin M. In metastatic lines the sensitivity to both cytokines is lost (Cancer Res,53:2708,1993). We hypothesized that in malignant melanomas there may be coregulation of response to lL-6 and the other cytokines which use the same gpl30 signal transduction system, namely, LIF and IL-11. Early-stage to advanced-stage melanoma lines (the WM8 series) were incubated with 0.001-100 ng/mL LIF and IL-11. No proliferative or inhibitory response to these cytokines was observed by ('H)-thymidine incorporation. LIF and IL-11 mRNA and protein expression was measured by reversetranscription polymerase chain reaction (RT-PCR) and bioassay from early-stage and advanced-stage melanoma lines. Whereas all the lines except one (MeWo) produced LIF mRNA and protein, only two lines (WM 793 and WM 983A) produced signiflcant IL-11 mRNA and protein despite the presence of smalt, but detectable amounts of IL-11 message in all the other lines. The lines secreting highest levels of LIF (WM 983), IL6 (MeWo) and IL-11 (WM 793) were different. No evidence could be found for either co-regulation of expression of these cytokines or the cellular responses of each individual line to them. However, there appears to be higher rates of mRNA and protein expression of LIF, IL-11 and IL-6 in the advanced-stage melanomas.
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PREDICTABLE CHANGES \N DIFFERENTIATION OF HUMAN MELANOMA CELLS HUMAN MELANOSOME SPECIFIC ANTIGEN (HMSA-6) IS ASSOCIATED WTffl THE INDUCED BY BROMODEOXYURIDINE AND MELANOCYTE STIMULATING HORMONE. EXPTJESSICW OF ER N E W D R K AND NEURAL CREST C3RANULES IN HUMAN MELANOCYTES GE Searles. H Chen. K Jimbow. Div.Derm.Cutan.Sci,U.of Albena, Edmonton, AB, AND MELANCMA CEIiS, H, Ctien. K, Jimbow. Div, of Dermatology & Cut:aneous Canada Sciences, University of Alberta, Eamonton, CANATA, The differentiation state of malionant celts correlates with metastatic potential in In order to characterize melanogenesis-associated proteins, we have many cell lines. Halogenated pyrimidine analogues like bromodeoxYuridine (BRDU); and recently developed several monoclonal antibodies, MoAbs, using melanoscmes growth hormones like a-Metanocyte Stimulating Hormone (a-MSH) can reversibly isolated from human melanocytes and melanoma cells. One of the recently established antibodies, MoAb HMSA-6 was found to be unique because it has change differentiation. We determined the effect of differentiation induction by these 4 antigenic epitopes with sizes of 11, 17, 22 and 41 kDa, that these 4 can agents on changes in morphology (cell size, shape, dendricity, melanosome number), cell proliferation, soft agar colony formation, and pigmentation on 4 non-metastatic be grouped into 2 species, (a) 41 kDa, and (b) 11, 17 and 22 kDa, after human malignant meianoma (HMM) cell lines, (WM35, WM39, WM902B, WM1341D) V8 protease digestion. This study has conducted the immunohistochemical and 4 metastatic HMM cell lines (SK-MEL23, SK-MEL24, G361, C32). ctiaiacteristics of MoAb HMSA-6 epitope distribution, using FITC labelled After 7 days in 1 8^M BRDU, all HMM, except WM1341D, were less differentiated indirect immunofluorescence and laser scan confocal microscc^jy, TVo patterns of immunofluorescence were seen in both normalroelanocytesand (25-99% less tyrosinase activity, reduced TRP-1 content, and melanin; 3-5x higher colony formation). However, WM1341D was more differentiated (3x more tyrosinase melaiKima cells, including amelanotic cells, Ihey were (a) reticular or activity and melanin; 3x fewer colony formations). No cell shape changes were filamentous network located in both perinuclear areas and dendrites, and observed, except WM1341D, which became 5x larger and highly dendritic. (b) round or granular perinuclear aggregates. Basically, similar findings After 7 days in 1O'^M a-MSH, all HMM, except WM35, were unchanged in all were obtained in human neuroblastoroa cells. In contrast, human fibroblasts revealed prinarily the fibrillar or reticular networks extending to the parameters compared to control. WM35 became more differentiated than control (lOx more tyrosinase activity, and melanin content; more dendritic). perikaryon and dendrite, T^e control specimen stained with antibodies against 10 nm vimentin filament and tubulin revealed entirely different We show that:1) BRDU and a-MSH can variably alter differentiation in all HMM; 2) patterns of filamentous network, indicating that HMSA-6 is not related to, WM1341 D becomes more differentiated with BRDU induction, while all other cell tines or associated with 10 nm vimentin or microtubules. We propose tiiat become less differentiated; 3) WM35 can become either more or less differentiated by filamentous or reticular pattem of HMSA-6 immunostaining is associated using a-MSH, or BRDU, respectively. WM35 and WM1341D could be very useful with the networks of endoplasmic reticulum and its granular perinuclear models for examining gene expression during differentiation in melanoma cell tines, deposits are related to neurosecretory granules common to both melanocytic especially WM35, since 1) it was derived from a radial-growth phase tumour, and 2) is cells and neuroblastoma cells. the most plastic of all cell tines tested with regard to differentiation.
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MEMBRANE "DIFFERENTIATION" IN STRATUM CORNEUM MEMBRANE MODELS. Neil KHson and Jenifer Thewalt, Division of Dermalology and Department of Physics , University of British Columbia. During epidermal differentiation, monoglucosylceramides are converted to ceramides by B-glycosidases. This lipid modification is one of a number that contributes to the formation of the stratum corneum interceilular membranes which are in turn responsible for the epidermal permeability barrier. We have previously shown by deuterium magnetic resonance and X-ray diffraction that modei membranes of composition simiiar to that found in the SC {i.e.. ceramide:chciesterol:palmitic acid 1:1:1 mol/moi) are composed of large crystalline domains co-existing with smaller fiuid domains at physiological temperatures, and we speculated that such architecture resulted In a porous medium through which diffusion may be described by percolation. In certain tissues (e.g., buccal mucosa) and disorders (Gaucher's Disease) the activity of (i-gtycosidases Is reduced or absent, and this is accompanied by a rate of transepithelial water loss that is greater than that observed in normal skin. We therefore examined model membranes composed of bovine brain monoglucosylceramide, cholesteroi, and perdeuterated palmitic acid (1:1:1 mol/mol) at pH 5.2. We found that solid domains were formed at room temperature, but that these were smaller than those found with ceramide, and transition to a fluid lamellar signals occurred at correspondingly lower temperatures. We believe that these results are consistent with the hypothesis that tissue properties, in this case transepidermai water loss, can be related directly to lipid paci
SPECTROSCOPIC AND MICROSCOPIC PROPI-RTIES Or SKIN AUTOFLUORl-SCENCE EMISSION. HaJRhan Zcnt;. Cnliim MacAulav. David 1. McLcnii*. llarvtfV Lui*. Orflnko Palcic. Cancer Imaging Department and *Division of Dcrmatoiogic Oncology, British Columbia Cancer Agency, Vancouver. To improve the understanding of human skin autofluoresccnce emission, the spectroscopic und microscopic charaeteristies of skin aulofluorcscencc were studied using a combined fluorescence and reflectance spectroanalyser and a fiber optic microspectrophotomcter. The aulofluoreseence spectra of in vivo human skin were measured over a wide range of" excitation wavelengths (350 nm - 470 nm). It was found that not only the intensity but also the sliape of these speetra were exeitation wavelength dependent. The excitation emission matrices (EEMs) of in vivo skin were constructed from these data. An exeitation emission maximum pair (380 nm, 470 nm) was identified. The most probable energy of skin autofluorescenee emission photons tends to increase linearly with increasing exeitation photon energy. We also demonstrated that the diffuse refieetanee (R) ean be used as a first order approximation of the fluoreseenee distortion faetor (/) to correct the measured in vivo autofluorescenee spectra for the effeet of tissue reabsorption and scattering. The microscopic in vitro autoOuorescenee properties of excised skin tissue sections were examined using 442 nm 1 ie-Cd laser light exeitation as an example. It was revealed that the fluorophore distribution inside the skin tissue is not uniform. The autofluorescenee speetra of different anatomical skin layers are also different. This study suggested that the major skin ttuorophores are located in the dermis. However, the exact fluorophore moiecuies have yet to be identified. Nevertheless, these mieroscopic data allowed us to reconstruct the 442 nm laser light exeited in vivo (macroscopic) autofluorescenee speetrum of skin tissue using Monte Carlo simulation.
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SKIN CYTOKINE PROFILES IN TIGHT SKIN (TSK) MICE WITH CD4 OR CD8 GENE DISRUPTION. Daniel N. Sauder. Takeshi Kono. Seiii Kondo. Savetia Pastore. Valerie A. Wallace. Christopher J. Paipe. Tak W. Mak and Roderick C. McKenzie. Division of Dermatology, Department of Immunology and Biophysics, University of Toronto, Toronto, Canada.
CD4 AND CD8 REQUIRED FOR OPTIMAL DEVELOPMENT OF ALLERGIC CONTACT DERMATITIS. Seiii Kondo. Gulnar M. Shivii. Takeshi Kono. Saveria Pastore. Tak W. Mak. and Daniel N. Sauder. Division of Dermatology and Department of Biophysics, University of Toronto, Toronto, Canada The use of gene targeting has provided powerful tool to examine precise functional roles of molecules in the immune system. In order to study the role of CD4 and CD8 molecules in the development of allergic contact dermatitis (ACD), the mutant mice lacking CD4 or CD8 were generated using gene targeted disruption. The mutant mice and syngeneie controls (C57BL/6) were sensitized on the abdominal skin with DNFB and challenged on the ears 6 days later. Ear swelling was assessed as a measure of ACD. Mice lacking CD4(-) had a marked hyporesponsiveness of 38.3±9.0% (P<0.025) in ear increment when compared with normal control. CD8(-) mice also showed hyporesponsiveness (67.8±7.0%, P<0.025) but to a lesser extent than CD4(-) mice. Skin.cytokine profiles after challenge were analyzed in CD4(-) mice by RT-PCR. Total RNA was extracted from the ear at various time points after hapten application to sensitized mice, reverse transcribed to cDNA, and amplified by PCR using labeled specific primers. Cytokine signal densities were expressed relative to p-actin signals and these ratios were compared to C57BL/6 controls. In normal mice, IFN-y, IL-6 and IL-10 mRNA were upregulated between 6-24 h after hapten application (42, 22 and 23fold, respectively at 24 h), while in CD4(-) mice these genes were not upregulated to the same extent (17, 17 and 15-fold, respectively). These results suggest that both CD4 and CD8 molecules are required for optimal induction of ACD. The CD4 molecule may influence the development of ACD by modulating the cytokine profiles in the skin.
The tight skin (Tsk) mice (dominant mutation in C57B1/6) represent a murine model of heritable fibrosis with some similarities to human scleroderma. To determine the role of T-cell subtypes in cutaneous fibrosis, the Tsk mice lacking CD4 or CD8 were generated using gene-targeting disruption. We have already shown that Tsk/CD4 (-) mice show less severe skin fibrosis compared with CD4(+)/CD8(+) or CD8(-) Tsk mice. In this study, we analyzed skin cytokine profiles by RT-PCR. Total RNA was extracted from Tsk, CD4 or CD8 gene-targeted Tsk and normal C57B1/6 mice, reverse transcribed to cDNA, and amplified by PCR using labelled specific primers. Cytokine signal densities were expressed relative to p-actin signals, and these ratios were compared to C57B1/6 controls (C57B1/6 ratio = 1 ) . In Tsk mice with severe fibrosis, TGF-P, bFGF and PDGF-A mRNA were upregulated (2.6 ± 0.4; 1.8 + 0.2 and 2.7 ± 0.2, respectively; P<0.05), whereas Tsk/CD4(-) mice with mild fibrosis had normal level of TGF-P and PDGF-A but suppressed level of IL-la, TNF-a and bFGF (0.5 + 0.1; 0.3 + 0.1 and 0.2 + 0.1, respectively; P<0.05). Tsk/CD8(-) mice had severe fibrosis but had normal levels of IL-la, bFGF and PDGF-A but TGF-P mRNA was elevated ((2.1 + 0.7, P<0.05). These results suggest that alteration in fibrogenic cytokines, especially TGF-P, is correlated with fibrosis in Tsk mice and that CD4 gene may influence fibrotic change in Tsk.
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INVERSE KfSTRDPHIC EPIDERMOLYSIS BULLOSA! REPORT OF 1W0 C3\SES WITH FURTHER OQEBEtATION BBIWEEN IMMUNOFLUORESCENCE STUDIES 2^ND ELECTRON MICBDSOOPX. A. Lin*. L.T. Smith**. J.D. Fine***. *The Rockefeller University, New York, NY. **University of Washington School of Medicine, Seattle, WA. ***University of North Carolina at Chapel Hill, NC. We present two patients with dystirophic EB whose lesions occurred in stir iking inverse distribution. A 17 year-old boy presented 4 years ago with blisters mainly in the groin, ankyloglossia, dysphagia and microstoroia which subsequently required plastic surgical reconst:ruction. Barium swallow showed proximal esophageal narrowing. Biopsy of a fresh blister showed separation below the lamina densa and diminished anchoring fibrils Oh electron microscopy, but n o m a l staining of LH 7:2 on immunofluorescence. A 9 year-old boy presented 4 years ago with blisters mainly in the axillae and groin, short frenulum and no dysphagia. Electron microscopy had previously shown separation below the lamina densa, and repeat biopsy showed sparse immature anchoring fibrils but normal staining with IH 7:2 antigen. These findings suggest that inverse dystrophie EB is a rare, unique subset characterized by normal M 7:2 staining but diminished anchoring fibrils, variable clinical course, and marked oral and esophageal involveinent in some patientis. It may possibly be caused by a point mut:ation in tiie type VII collagen gene at a site whose ej^tession is not detected by the monoclonal antibody LH 7:2.
LOCALISATION DES ANTICORPS ANTI-MEMBRANE BASALE DANS LA PEMPHIGOIDE. B. Mavnard. Service de Dermatoloyie, Di5partement de M^ecine, Centre Hospitaller Universitaire, UniversitiS de Sherbrooke, Sherbrooke. Leu maladies du groupe de la pemphigoide et celles du groupe de I'^pidermolyse bulleuse acquise (EBA) peuvent se traduire par un tableau clinique et histopathologique semblable et par une immunopathologie de routine identique. La technique du clivage de la membrane basale par le NaCl permet de s6parer physiquement les antig&nes de la pemphigoide (composants des hdmidesmosomes, retrouvds du cfit^ ^pidermique de la separation) de I'antigfene de I'EBA (le collag&ine de type VII constituant des fibrilles d'ancrage, retrouv^ du c6t^ dermique de la siSparation). L'immunofluorescence indirecte sur ce substrat a permis de caractdriser les anticorps circulunts contenus dans les scrums de 7 patients avec pemphigoide bulleuse et 2 patients avec pemphigoide cicatricielte. Tous les scrums testes (100%) ont montr6 un patron ^pidermique (toit de la buUe). Le titre des unticorps circulants fut determine sur oesophage de singe et sur peau humaine dissociee dans 6 cas. Dans tous les cas le titre titait plus 6leviS sur In peau dissociiSe, La technique de I'^piderme dissoci^ est simple et permet de caracttSriser les anticorps circulants dans la pemphigoide. La prevalence de I'EBA est faible dans notre milieu. La peau dissoci^e est un substrat plus sensible que l'oesophage de singe pour diStecter des anticorps circulants.
ABSTRACTS
VOL. 103, NO. 2 AUGUST 1994
143 T TUMOR INCIDENCE AND DNA REPAIR IN UV EXPOSED P53 TRANSGENIC MICE G Li''. D Mitchell. VC H(i- and VA Tron'-. Du partment of TRANSGENIC Division MICE. ol Dermatology^, University of British Columbia, BC und M,D. iii P tholoijv' Anderson Cancer Center'' University of Texas, Texas. n53 is an important tumor -suppressor, p53 protein is thought to regulate the cell cycle after a DNA damage event. Mutations ot p53 geno have been implicated in the oathocenesis of cutaneous squamous cell carcinoma (SCC), To examine the role of p53 in skin carcinogenesis, we observed the development of skin cancers in p53 transjjenic mice, which overexpress mutant alleies of the p53 gene, and control mice with or without UVB for 34 weeks. 28 tumors developed in 19 p53-UV irradiated mice versus 14 of 18 in the control vroup- 9 p53 niice but none of the control mice developed multiple tumors. Histologically, most tumors were spindle cell malignancies, consistent with spindle cell squamous cell carcinomas. The mean time to appearance of a tumor (minimum of 5mm in diameter and histologically proven malignancy), did not difter between the two groups (p = 0.16). To determine whether the presence of p53 mutations alters DNA repair, cyclobutane diniers and 6-4 photoproducts were assayed in skin after exposure to UV radiation and expressed as % remaining photoproduct post radiation. The p53 transgenic animals demonstrated decreased repairof cyclobutane dimers at 4 hrs (control 55±9 vs. p53 103±6, p = 0,0002*) and 24 hrs fcontrol 56 + 11 vs. p53 77ji7, p = O.OOI *), and reduced repair of 6-4 photoproducts at 4 hrs (control 3 5 ^ 8 vs. p53 85±7, p = 0.0001*). Our findings indicate that mutant p53 predisposes mice to multiple and increiised numbers of UV-induced tumors, but does not shorten the latency period for tumor development. Multiple and increased numbers of tumors in p53 transgenic mice may be the result of reduced DNA repair. *(meanJiSEM)
145 TRO CYTOTOXICiTY OF PHENOLIC THIOETHERS. A.F-. Gili, M, Tiincion, T, ~K. Jinihow. University of Alberta. Division of Dermatology and Cutaneous Sciences. Edmonton. Alberta, We have recently synthesized two new phenolic thiocthcrs in our laboratory, N-propii)nyl-4-Scysteaminyl phenol (NPr-4S-CAP) ;ind N-{2-|(4-propionyloxypheny!)thio]etliyl}propioiunnidc fNPP-TEP) These compounds are tyrosine analojjucs, similar to other previously deseribed phenolic thiocthers such as 4S-cysteaminyl phenol (4S-CAP) ;ind N-acetyl-4S-cyte:iminyl phenol (NAC-4S-CAP). The cylotoxieity of these latter two compounds have been proposed to be primarily tyrosinasc dependent, whieh may make them suitable as specific anti-metanonia therapy sinee only melanocytes and their malignant counterparts ctintain this active enzyme. To further investigate this hypothesis, we preformed MTf (3-(4.5-diiTieihylthiazol-2-yl)-2.5diphenyltetrazolium bromide) assays as a method of determining cytotoxiciiy on 9 different cell lines using the NPr-4S-CAP and NPP-TEP compounds. We also performed the same assays using NAe-4S-CAP and a pro-drug, N-{2-[(4-acetoxyphenyl)thiojethyl}acetaniide (NAP-TEA), which was also developed in our laboratory. The cells were exposed to the drug for two days. Of the nine cell lines, all the human neuroblastoma lines (SK-NMC. SK-NSH, KAN) and an amelanotic human melanoma line (C32) were consistently most sensitive to all the drugs. The consistently most resistant cell lines included SK-MBL-23 (highly pigmented human melanoma), SK-MEL-24 (amelanotic human melanoma) and human fibroblasts. The eell lines in the middle range of sensitivity were HeLa (human cer\'ieal eanccr) and G.Vji (human melanoma with low tyrosinase activity). Our present in viiro study may indicate ihat there is a non-tyrositiase mediated eytotoxicity for phenolie thioethers in addition to the lyrosinase mediaicd one as previously proposed.
147 MANGANESE INDUCES INTEGRIN-MEDIATED HUMAN MELANOMA ATTACHMENT AND SPREADING ON LOWER RGD DENSITIES ON DEFINED SUBSTRATES. GE Searles. K Jimbow.Div.Derm.Cutan.Sci,U. of ALberta, Edmonton, AB, Canada Glv-Arg-Gly-Asp-Ser (GRGDS) is a ligand for several integrins expressed on human malignant melanocytes, including tha metastatic malignant melanoma cell line, G361. To determine the effect of cations on the minimal number of ligand-receptor interactions required for G361 adhesion, GRGDS was covalently grafted at densities ranging from 0,1 to 10* fmol/cm^ to poorly adhesive glass substrates, Cell morphologies were determined in serum-free media (SFM) with or without manganese (Mn'^1 present. Perturbation of adhesion by functional anti-integrin antibodies was determined, as was the sensitivity of adhesion to several cytoskeletal inhibitors. G361 adhesion to GRGDS was divalent cation-dependent, and increased with increasing GRGDS densities. Adhesion was greater with Mn"^ (1 mMl compared to SFM at each GRGDS density. Optimal cell spreading occurred at an RGD density of 10 fmol/cm' with SFM, and at 1 fmol/cm* with Mn"^ present. With SFM, adhesion was inhibited with anti-a3 (76%) and anti-Bl (97%) antibodies; with M n ' ^ anti-a2 (34%), anti-CT3 (90%), anti-o5 (15%) and anti-GI (93%) antibodies were inhibitory. Increased membrane viscosity (paraformaldehyde) had equivalent inhibition (70%) in both cation conditions; whereas actin depolymerization (cytochalasin D), Integrin-cytoskeletal unlinkage (dibucalne) and glycolysis inhibition (sodium fluoride) was more inhibitory for G361 cells with Mn'^ present, than with SFM alone. We show that; 1) adhesive peptides can be covalently grafted in a quantitative manner; 2) G361 cells can bind to this substrate in a divalent cation-dependent fashion; 31 manganese permits adhesion at lower RGD densities, probably through recruitment of o2B1 and o5fi1 integrins and increased cytoskeletal organization. This fully-defined substrate will be useful In precise quantification of each component of the adhesion cascade.
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144 TISSUE DISTRIBUTION OF UV-INDUCIBLE mRNA TRANSCRIPTS IN THE ADULT MOUSE C. Rosen. R. Poon. R. Rathee. D. Drueker. Department of Medicine, Women's College Hospital and Toronto General Hospital, University of Toronto, Toronto. We have recently reported the isolation of 23 keratinocyte mRNA transcripts that were induced by ultraviolet B radiation in neonatai rat keratinocytes in vitro. To determine whether the novel UVB-induced genes were also expressed in non-cutaneous tissues in vivo, we have isolated RNA from a variety of Skh-1 hairless mouse tissues, and characterized the tissue distribution of gene expression for 15 of the 23 genes in wVo using Northern blot analysis. Several distinct patterns of gene expression were identified. Ali of the genes were expressed in mouse skin. Two genes were strongly expressed in skin with only minimal expression detected in other tissues. Four genes were expressed in all tissues studied, pancreas, liver, spleen, kidney, small and large bowel, lung, heart and skin. Two genes were expressed oniy in the skin and the gastointestinai tract. Generally, the pancreas showed the lowest level of transcript expression and four genes were expressed in ali tissues except the pancreas.These observations demonstrate that genes originally isolated from a keratinocyte cDNA library are generally expressed in a diverse number of mouse tissues, suggesting that the proteins encoded by the majority of these UVB-induoed cDNAs likely subserve distinct ceiiuiar functions not restricted to the biology of the keratinocyte In vivo.
146 BIOUOGIGRL ROLE OF TYPOSINASE-REIATED PROTElN-1 (TRP-1) DJ MEIANDGENESIS' IN CtHTRANSFECTED CELLS OF HUMAN TYRDSINASE AND TRP-1 GENE. K. Jimbow, D. Ijuo. H. Chen, Div. of Deniiatolagy & Cutaneous Sciences, University of Alberta, Edmonton, CANADA, Tyrosinase is a key enzyme responsible for melanogenesis. Recently however, 2 tyrosinase-related proteins (TRP-1, 2) and encoding genes have been identified. Tt^P-2 has been attributed to dopachrome tautomerase which converts dopachrome to dihyrdoxindole 2 carboxylic acid to fortn eumelanin. In contrast, the nature of TRP-1 is still unclarified. In order to characterize the possible biological role of TRP-1 in melanogenesis, we constructed 2 genes specific to melanogenesis, hunan tyrosinase (HT) and TRP-1, into monkey kidney oos-7 cells and human amelanotic melanoma cells. Transfectants were examined for mRNA expression by reverse transcription mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectant or co-transfectant. Both light and electron microscope observation indicated that degeneration and premature death of melanocytes occurred by HT transfection, but not by TRP-1 alone or by combination of both. Co-transfected cells from amelanotic melanoma cells and COS-7 cells revealed a granular product which reacted with a specific anti-TRP-1 antibody and exhibited numerous lysosomal vacuoles with electron-dense material, indicating aggregation/degradation of new protein product, as well as melanin in lysosomal vacuoles. Our melanin assay confirmed new production of melanin in these cells. There was also a dranatic elevation of gene and protein expressions of lanp-l (lysosomeassociated membrane protein-1) in co-transfected cells. TRP-1 gene may, therefore, f unct ion together with Lairp-l by stabi 1 iz ing the enzymic conplex of tyrosinase in melanogenesis in order to have proper sequence of melanogenesis events as well as preventing tyrosinase-mediated cytotoxicity.