Poster Presentations
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GENETIC AND IMMUNOCHEMICAL ANALYSIS OF MUTANT P53 IN HUMAN MALIGNANT MELANOMA CELL LINFS. Yukio Yamashina. Haruhiko Ohno, Masami Yamaii, Nobuhiko Kobavashi. Tsutomu Muramatsu, Toshihiko Shirai, Hideki Matsumoto’ , Kumio Okaichi” and Masamitsu Ichihashi”’ Department of Dcrmatoloev. 1st Dcoanment of Anatomv’ and Deoattment of Bioloev”. Nara Medical U&crsity, I&shihara and Depart&ent of De~atology, Kobe U&enity”‘, Kobc, Japan Recently, the expression of the tumor suppressor gcnc pS3 is analyzed in various human malignant tumors and the mutation of the ~53 gene is reported in many tumors. Tbcn the expression of the pS3 protein in various malignant skin tumors is also reported. In the prcscnt study, WF examined the mutation of ~53 genetically and immunochcmically in 6 human malignant melanoma cell lines (HMVl, HM6K0, G361, P39, I’22 and Mewo). In Western blotting analysis using an anti-p53 moooclonal antibody (PAbl801) with BLAST system for amplification, HM6KO showed the strong immunorcactant but other CLII lines did not show positive reaction. For sequencing analysis of genomic DNA from these 6 melanoma cell lines, the regions exon 5 to 9 of the ~53 gene were amplified into 3 fragments. From the analysis of the PCR products, we did not detect the homozygous dclction of the exons. Therefore, it is suggested that HM6KO has mutation of the ~53 gent and synthesizes abnormal ~53 protein.
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va,Y.Kohno. Dept. Cancer
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Spr genes were originally isolar.& from a htman epidermal cDN.4 library as IN-inducible IT. Kartasova and P. van de Mol. Cell BL01.8: 2195-2203, 1988). Subsequently, it was shown that the expression of the*e genes is also induced
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by TPR and during epidermal differentiation. Mouse homologues of human spr genes were isoLated 11 for the experimental carcinogenesis studies in a mouse skrn model; 2) to get insight on the evolution of the*e genes in the animal kingdom. Complete sprI and sprI* cDNA* were isolated from a mouse papilloma cDNA library. Sequence comparison *howed that the mm*e spr genes were 60% larger than their human homologue* due to a larger number of the conserved repeats. The overall str”cture was preserved between mause and human spr genes. Northern blot analysis of RN* isolated from mouse embryonal skin revealed induction of the spr gene expression on day 16 which correlated closely with the induction of stratification in fetal skin. Antibodies were raised in rabbits using synthetic peptides corresponding to the N- and C-termini of the s~r1 and snrII oroteins and nurifled bv afflnltv chromatography: ThL specificity’of these antibodies is currently being tested by weStern blatting and by immunohistochemical staining using mouse and human samples.
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DISTRIBUTION OF POLY-A mRNA IN NORMAL HUMAN SKIN. Takuji Masunagat.3. Shin-&hi Takahashiz, Hiroshi Shlmizo’ and Takep Nishikawa’, tDeoartment of Dcrmatotoev. Keio Universitv School of Mediane. Tokvo. %epartment of Dermatology. lchikawa General Hospital of Tokyo Dental College, Chiba. )Research Laboratory. KOSI? Corporaoon. Tokyo, Japan. The amount and distribution of mRliA may reflect the cell actwity To mvestigate the distribution of mRNA in the skin is ortportant to clarify the skm biology and pathophysiology. However, there are few reports concerned with the distribution of poly-A mRNA in the skin. and its distribution is not yet established In this study, we attemptedto find out the optimal condition to demonstrate the distnbution of @y-A mRNA in normal human skin by in ritu hybndization. Normal human skin was snap frozen and embedded in OCT compound, and stored in liquid nitrogen until use. The b pm-thick sections were fixed in 4% parafotmaldehyde. After various pretreatmcnts were applied. the sections were hybridized with digoxigenin labelled poly-dT (3Omer) probe. Hybridized probe was immunohisto&emieaIly detected by usmg sheep anlidlgoxtgenin antibody conjugated to alkaline phosphatase. As a pretreatment! a combination of proteinase K (33 ngiml. 15 min) and acetylation wth acetic anhydnde (0.25 v/v%. I5 min) gave the highest signal noise ratio. In all epldermal cells including granular, spinous and basal layer, the positive signal was equally observed in the nucleus and perinuclear space. The nucleus and cytoplasm of skin appendages were stained more strongly than those of epidemnl cells. The all signals were completely disappeared by RNase treatment. Contrary to the previous report that a gradtent of stgnal was observed with low amounts in basal layer. our results indvzate that poly-A mRNA is expressed uniformly in the entire epidermis and expressed more strongly in the skin appendages. The distribution pattern which is different from that to other tissues mdacates that the slun might undergo different regulation from other t~ssees.
POSITIVE AND NEGATIVE ELEMENTS INVOLVED IN THE TRANSCRIPTION OF THE HUMAN PROFILAGGRIN GENE. S. lane, N.G. Markova. and PM. Steinett, Skin Bioloev Branch. NIAMS. NIH. Bethesda. Matvland. U.S.A. H&an proiilaggrin ‘is a major ep&&l i&mediate filament-associated protein. It is synthesized in the late stages of epidemml differentiation. Tbe level of expression is calcium dependent, and in cultured keratinocytes, profilaggrin mRNA tranxription can be induced wtth calcium. Recently we characterized the human profilaggrin transcription unit and especially the 5’ region where most of the regulatory signals are normally located. The proximal and distal promoter regions co&n tiny reguIat&y elements, some of which have been implicaied in the regulation of other epidermally expressed genes. To eluddate the regulation of transcription of profilaggrin mRNA, a series of fragments encompassing gradually increasine cations of the S-reeion of the orofilaeeti eene were cloned into a CATreporter vector and used in a t&dent e&ssion&ay~The CAT expression was low in epithelial cells (HeLa) and undifferentiated epidermal cells, such as RHEK and cultured normal human epidennal keratinocytes (NHEK) grown in low calcium. When calcium concentration was raised to 1.2 mM in NHEK, different degrees of CAT expression were observed fmm each construct. Similar profiles of CAT expression were found in HaCat cells. The ~onstrwt containing first 120 bp upstream of the transcription initiation site exhibits a significant enhancer activity. Bandshift experiments with NHEK and HaCat cellular extracts reveal that at least three proteins bind to regulatory sequence sites in this proximal promoter region confirming a differentiation specificity to the expression. An enhancer element Is situated between positions -359 and -705. Three negative elements are located between positions -120 and -359, -705 and -1500, and -1500 and -3&X, respectively. Further band shift experiments and DNA footprinting analyses will now provide a detailed characterization of these regulatory sequences.
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