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-183- Cell Biology/General 9/5 Room-D
IDENTIFICATION OF GENES WHICH SHOWS SPECIFIC EXPRESION IN DEVELOPING MOUSE SKIN.
Nam-ho, Huh1, Mikiro Takaishi1, Hiroaki Kinoh1, Yoshimi Takata1, Gabriela Yamago2.
1Department of Biochemistry, 2Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.
In order to reveal molecular mechanisms of morphogenesis of skin, we studied the specific expression of genes in developing mouse skin on 12 to 16 gestational days. 1) By RNA differential display, we have identified 11 novel gene fragments, whose expression levels were up- or down-regulated in the process of morphogenesis. 2) Sonic hedgehog, patched, jagged, Notch-1, EGF receptor and its ligands were differentially expressed, indicating their involvement in the morphogenesis. 3) By RT-PCR cloning, 23 among 38 known Hox genes were identified in the dorsal skin of 13-day embryos.
-184- Cell Biology/General 9/5 Room-D
EXPRESSION OF WNT GENES IN MURINE EPIDERMIS.
Atsushi Saitoh1, Shinji Shimada1, Mark C.Udey2.
1Department of Dermatology, Yamanashi Medical University, Yamanashi, Japan. 2Dermatology Branch, NCI, NIH, Bethesda, MD, USA.
Murine Wnt gene productsplay important roles in morphogenesis and tumorgenesis, and mayhave many additional functional activities. As a prelude to study of wnt genes as regulators of skin development and function, we characterized wnt gene expression in murine epidermis. Degenerate primers complementary to conserved regions wnt cDNAs and RT-PCR were used to amplify wnt-related sequences that were present in adult C57BL/6 epidermal total RNA. PCR products of appropriate size were cloned into pBluescript, 23 clones were analyzed by limited restriction mapping, and unique clones were sequenced. The most frequently recovered partial cDNA sequneces corresponde to wnt-4, although other wnt-related sequences were also identified. Presence of wnt-4 mRNA in epidermal RNA was confirmed by RT-PCR and RNase protection assay ( RPA ). Furtheremore we investigated the wnt-4 function by using wnt-4 knockout mouse.
-185- Cell Biology/General 9/5 Room-D
SKEWED METHYLATION OF X CHROMOSOME IN NORMAL SKIN OF VARIOUS AGES.
Hiroshi Fujiwara, Masaaki Ito.
Department of Dermatology, Niigata University Scool of Medicine, Niigata, Japan.
Skewed methylation of X chromosome in female is considered as a sign of monoclonal cell expansion in a malignant disease. Generally the ratio of two X chromosome methylation by 7:3 or more is regarded as 'skewed', however, methylation analysis in normal skin have not been performed, so far. We performed methylation analysis of androgen receptor gene, with DNA extracted from normal skin of ages 0-10, 21-30, 61or more. The DNA was pretreated with or without methylation-sensitive restriction enzyme, amplified, run on polyacrylamide gel, and silver-stained. 3/20, 2/18, 5/15 of each age-group revealed skewed pattern, respectively. In the normal skin the methylation pattern can be skewed, and it can occur more likely in old ages.
-186- Cell Biology/General 9/5 Room-D
IDENTIFICATION AND ANALYSIS OF 40 KDa PROTEIN LOST IN TUBEROUS SCLEROSIS.
Mari Wataya-Kaneda1, Yasufumi Kaneda2, Kunihiko Yoshikawa1.
1Department of Dermatology, Osaka University School of Medicine, 2Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan.
Tuberous sclerosis is a genetic disease characterized by systemic hamartomas. Some of the cultured cells derived from tuberous sclerosis(TS cells) show the distinct chromosomal disarrangement and abnormal cell division. As reported previously,40 kDa protein (p40), which was present in nucleus, chromosomes and cytoplasms of many vertebrate culture cells, significantly decreased in TS cells. In this time, we isolated the p40 from cytoplasmic particles, and identified it by extensive microsequencing as LBP-p40, which was considered to be a precursor of laminin binding protein p67(LBP-p67). Western blotting analysis using a polyclonal anti-LBP antibody revealed the loss of not the LBP-p67 but the LBP-p40 in TS cells. Ratio of LBP-p40 and LBP-p67 ( LBP-p40 / LBP-p67) also decreased in TS cells. Northern blotting analysis revealed that the transcriptionals of LBP-p40 was also inhibited in some TS patients, while it was not affected in other patients. Thus, in TS patients, the expression of LBP-p40 was supressed either in transcriptional level or in translational level.
-187- Cell Biology/General 9/5 Room-D
MULTIPLE CADHERIN EXPRESSION IN HUMAN FIBROBLASTS.
Norihisa Matsuyoshi, Sadao Imamura.
Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Although the cell-cell adhesiveness of fibroblasts is thought to be related to wound healing, the molecular basis of this adhesiveness is still unknown. We isolated five kinds of cadherin fragments from the cDNA of human fibroblasts by PCR. Two of the five were known cadherins: PC43, a protocadherin containing six cadherin repeats, and human Fat, which is the human homologue of the Drosophila tumor suppressor Fat. The other three were novel cadherin fragments, and we named them cadherin FIB1, FIB2, and FIB3. The expressions of cadherins including E-, P- and N-cadherin, PC43, human Fat, and cadherin FIB1, FIB2, and FIB3 were compared in fibroblasts, melanocytes and keratinocytes. The latter six cadherins were expressed in human fibroblasts, and cadherins FIB1 and FIB2 were fibroblast-specific. These results suggest that diverse cadherin molecules may contribute to the cell-cell adhesion in human fibroblasts.
-188- Cell Biology/General 9/5 Room-D
EXPRESSION OF CADHERIN-CATENIN COMPLEX IN HUMAN LEUKEMIA CELL LINES.
Kyoko Kawamura1, Jun-ichiro Tsutsui2, Masayuki Ozawa2, Tamotsu Kanzaki1.
1Department of Dermatology, 2Department of Biochemistry, Kagoshima University Faculty of Medicine, Kagoshima, Japan.
Cadherins are Ca2+ -dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the histo-architecture. Using cultured human leukemia cell lines (adult T cell leukemia and thymus derivide lymphoma cell lines), we obteined evidence that cadherins and catrenins were expressed in these cell lines but not in normal leukocytes. Sequencing of the cDNA fragment isolated from the cells revealed N-cadherin, E-cadherin, R-cadherin, P-cadherin, cadherin-11,and a newly found cadherin. Immunoblot analysis revealed the presence of N-cadherin and R-cadherin. Northern blot analysis revealed the presence of N-cadherin,R-cadherin and cadherin-11. Given the importent role for cadherins in cellular adhesion and signaling, expression of cadherin-catenin complex portends an importent role for these molecules in the behavior of the leukemia cells.