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183 Identification of genes which shows specific expres ion in developing mouse skin
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181 ENBENCE OF CULlURED KERATlNDCYlES AND THE EXPRESSION OF P21. K. Sayama, Y. Shirakata, K. Midorikawa, Y. Hanakawa, K. Hashimoto. Dsoartment of Dermatolopv._. Ehime University School of Mediclne, Ehime, Japan. The protein p21 is an inhibitor of cyclin-dependent kinase and We have studied affects cell cycle progression and ssnascsnce. whether p21 was associated with cultured keratinocytes sanascenca. Keratinocytes wars isolated from normal human skin obtained from individuals agsd from 1 to 70 years, and cultured under serum frse condition. The cultures have been continued until the call growth arrest. The expression of p21 in the growing calls and the sanascanl calls was anah/zsd by Western blot. Tha keratinocytes obtained from younger lndivlduals tend lo survive longer periods than the calls from older persons. The p21 expression has not been char!@ compared lo the early passags while the calls wars growing, but Increased just before the growth arrest. These results suggest that p21 might play an important role in growth arrest of ksratinocyts senescence.
182 ESTABLISHMENT 0~ 4 SPONTANEOUSLY IMMORTALIZED HUMAN KERATINOCYTE CELL LINE FROM NORMAL ADULT SKIN. T. Karashima , 0. Mori , T. Hashimoto. Department of Dermatology, Kurume University School of Medicine, Fukuoka , Japan. Spontaneously immortalization of human, keratinocyte has We have been reported, although such reports are still very few. established the spontaneous immortalized of human keratinocytes from normal adult skin. This Kana cell line is established from a long-term primary culture of epidermal keratinocytes grown in DMEM contained 10% FCS. These cells can be considered immortalized (>iOOpassages), and exhibit karyotypical abnormality, In low Ca++ m&urn, the cells predominantly grew as monolayers. However in high Ca++ medium, the cells readily This cell line may be stratified and formed coherent horn sheets. a useful model for the study of human keratinocytes.
183
IDENTIFICATION OF GENES WHICH SHOWS SPECIFIC EXPRESION IN DEVELOPING MOUSE SKIN. N. Huh, M. Takaishi, H. Kinoh, Y. Takata, G. Yamago. Department of Biochemistry, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Medical and Pharmaceutical University, ToyaIM Toyama, Japan. In order to reveal molecular mechanisms of we studied the specific morphogenesis of skin, expression of genes in developing mouse skin on 12 to 16 gestational days. 1) By RNA differential display, we have identified 11 novel whose expression levels Were upgene fragments, or down-regulated in the process of Sonic hedgehog! patched, morphogenesis. 2) Notch-l, EGF receptor and Its ligands jagged, were differentially expressed, indicating their involvement in the morphogenesis. 3) By RT-PCR cloning, 23 among 38 known Box genes were identified in the dorsal skin of 13-day embryos.
133
184 BXPRBSSIONOFWNTGENESINMURINE BPIDBRMIS. A.Saitoh*'.S.Shimada', M.C.Udev'. ' DqmrtmentofDarmatology, YamanashiMedicalUniversity.Yamanashi,Japn."Dermatdogy Branch,NCI,NIH,Bethesda,MD,USA. Murine Wntgane products play important roles in morphogcnenia
and
tumorgenesis, and may have many additional functional a&it&s. As a prelude lo study of wnt genes as regulators of skin development and
runction, we. charac~rized writ gene expression in murina epidamis. Degenemteprimers wmplameniary to conserved regions wnt cDNAs and RT-PCRwere used tc amplifywnt-related sequences Ihat werepresent in aduitCS7BU6epidarmaltoIalRNA. PCRpmduas ofappm@tasizewen cloned into pBlueacript, 23 clones were analyzed by limited r&icIion mapping, and unique clones were sequenced. The most frequently mcovacd partial cDNA sequneces axresponde to writ-4, although other wnt-related sequences were also dentified. Presence of wnt4 mRNA in epidermal RNA was confirmed by RT-PCR and RNase protztion assay ( RPA ). Furtheremore. we investigated the wnt4 function in murinc skin by using wnt-4 knockout mouse
185 SKEWED METHYIATION OF X CHROMOSOME IN NORMAL SKIN OF VARIOUS AGES. H.a’. M. lip. Department of Dermatology, Nilgata University School 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 73 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 O-10, 2130, 61or more. The DNA was pretreated with or without methylation-sensitive restriction enzyme, amplified, run on polyacrylamide gel, and silver-stained. 3I20, 2/l& 5115 of each age-group revealed skewed pattern, respectively. In the normal skin the methytatlon pattern can be skewed, and it can occur more likely in old ages.
186 IDENTIFICATION ANDANALYSIS OF 40 KDa PROTEIN LOSTIN TUBEROUS 7 1 SCLEROSIS. p ’ ‘Department of Dermatology, Dsaka Univarslly Schoolof Msdldna, Osaka, Japan. ‘lnslihde for Molecular and Cellular Biology, DsakaUnhrsrslty, Dsaka, Japan. Tubemus sclsrosis is a ganallc disease characiatfzad ty systamk hamarlcmaa. Sane of lhs cultured calls derived from tubemus scbmsts(TS calls) show the distinct chromosomal disarrangement and abnormal call dMsion. As repotted previously,40 kDa pmleh (p40), which was fxasam In nuciaus, chromosomes and cyIoplasms of many vm culture cells. signlfkant& dacraasad in TS calls. In lhia time, we isolated the p40 from cymplasmk partkks, and identlfkd it w extanslve microsequendng aa LBP-p40, which was mnsk%rad lobs a precursor of latinin binding prolain pG’(LBP-pS7). Waslam Molting an&y&s using a polydonal anti-LBP antibody revealed the loss of not the LBP-pS7 but the LBP-p40 in TS cells. Ratio of LBP-p40 and LBP-p67 ( LBP-p40 I LBP-ps7) also dacraasad in TS calls. Northern Molllng anatysis revealed that the tmnscriptionafs of LBP-p40 was also inhibited h some TS patlsnls, while ll was not affacted in other paIients. Thus, in TS patlsnls. the expression of LBP-p40 wassuprassad silhar in transctiptbnal level or in translatIonal Ibvel.
181 ENBENCE OF CULlURED KERATlNDCYlES AND THE EXPRESSION OF P21. K. Sayama, Y. Shirakata, K. Midorikawa, Y. Hanakawa, K. Hashimoto. Dsoartment of Dermatolopv._. Ehime University School of Mediclne, Ehime, Japan. The protein p21 is an inhibitor of cyclin-dependent kinase and We have studied affects cell cycle progression and ssnascsnce. whether p21 was associated with cultured keratinocytes sanascenca. Keratinocytes wars isolated from normal human skin obtained from individuals agsd from 1 to 70 years, and cultured under serum frse condition. The cultures have been continued until the call growth arrest. The expression of p21 in the growing calls and the sanascanl calls was anah/zsd by Western blot. Tha keratinocytes obtained from younger lndivlduals tend lo survive longer periods than the calls from older persons. The p21 expression has not been char!@ compared lo the early passags while the calls wars growing, but Increased just before the growth arrest. These results suggest that p21 might play an important role in growth arrest of ksratinocyts senescence.
182 ESTABLISHMENT 0~ 4 SPONTANEOUSLY IMMORTALIZED HUMAN KERATINOCYTE CELL LINE FROM NORMAL ADULT SKIN. T. Karashima , 0. Mori , T. Hashimoto. Department of Dermatology, Kurume University School of Medicine, Fukuoka , Japan. Spontaneously immortalization of human, keratinocyte has We have been reported, although such reports are still very few. established the spontaneous immortalized of human keratinocytes from normal adult skin. This Kana cell line is established from a long-term primary culture of epidermal keratinocytes grown in DMEM contained 10% FCS. These cells can be considered immortalized (>iOOpassages), and exhibit karyotypical abnormality, In low Ca++ m&urn, the cells predominantly grew as monolayers. However in high Ca++ medium, the cells readily This cell line may be stratified and formed coherent horn sheets. a useful model for the study of human keratinocytes.
183
IDENTIFICATION OF GENES WHICH SHOWS SPECIFIC EXPRESION IN DEVELOPING MOUSE SKIN. N. Huh, M. Takaishi, H. Kinoh, Y. Takata, G. Yamago. Department of Biochemistry, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Medical and Pharmaceutical University, ToyaIM Toyama, Japan. In order to reveal molecular mechanisms of we studied the specific morphogenesis of skin, expression of genes in developing mouse skin on 12 to 16 gestational days. 1) By RNA differential display, we have identified 11 novel whose expression levels Were upgene fragments, or down-regulated in the process of Sonic hedgehog! patched, morphogenesis. 2) Notch-l, EGF receptor and Its ligands jagged, were differentially expressed, indicating their involvement in the morphogenesis. 3) By RT-PCR cloning, 23 among 38 known Box genes were identified in the dorsal skin of 13-day embryos.
133
184 BXPRBSSIONOFWNTGENESINMURINE BPIDBRMIS. A.Saitoh*'.S.Shimada', M.C.Udev'. ' DqmrtmentofDarmatology, YamanashiMedicalUniversity.Yamanashi,Japn."Dermatdogy Branch,NCI,NIH,Bethesda,MD,USA. Murine Wntgane products play important roles in morphogcnenia
and
tumorgenesis, and may have many additional functional a&it&s. As a prelude lo study of wnt genes as regulators of skin development and
runction, we. charac~rized writ gene expression in murina epidamis. Degenemteprimers wmplameniary to conserved regions wnt cDNAs and RT-PCRwere used tc amplifywnt-related sequences Ihat werepresent in aduitCS7BU6epidarmaltoIalRNA. PCRpmduas ofappm@tasizewen cloned into pBlueacript, 23 clones were analyzed by limited r&icIion mapping, and unique clones were sequenced. The most frequently mcovacd partial cDNA sequneces axresponde to writ-4, although other wnt-related sequences were also dentified. Presence of wnt4 mRNA in epidermal RNA was confirmed by RT-PCR and RNase protztion assay ( RPA ). Furtheremore. we investigated the wnt4 function in murinc skin by using wnt-4 knockout mouse
185 SKEWED METHYIATION OF X CHROMOSOME IN NORMAL SKIN OF VARIOUS AGES. H.a’. M. lip. Department of Dermatology, Nilgata University School 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 73 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 O-10, 2130, 61or more. The DNA was pretreated with or without methylation-sensitive restriction enzyme, amplified, run on polyacrylamide gel, and silver-stained. 3I20, 2/l& 5115 of each age-group revealed skewed pattern, respectively. In the normal skin the methytatlon pattern can be skewed, and it can occur more likely in old ages.
186 IDENTIFICATION ANDANALYSIS OF 40 KDa PROTEIN LOSTIN TUBEROUS 7 1 SCLEROSIS. p ’ ‘Department of Dermatology, Dsaka Univarslly Schoolof Msdldna, Osaka, Japan. ‘lnslihde for Molecular and Cellular Biology, DsakaUnhrsrslty, Dsaka, Japan. Tubemus sclsrosis is a ganallc disease characiatfzad ty systamk hamarlcmaa. Sane of lhs cultured calls derived from tubemus scbmsts(TS calls) show the distinct chromosomal disarrangement and abnormal call dMsion. As repotted previously,40 kDa pmleh (p40), which was fxasam In nuciaus, chromosomes and cyIoplasms of many vm culture cells. signlfkant& dacraasad in TS calls. In lhia time, we isolated the p40 from cymplasmk partkks, and identlfkd it w extanslve microsequendng aa LBP-p40, which was mnsk%rad lobs a precursor of latinin binding prolain pG’(LBP-pS7). Waslam Molting an&y&s using a polydonal anti-LBP antibody revealed the loss of not the LBP-pS7 but the LBP-p40 in TS cells. Ratio of LBP-p40 and LBP-p67 ( LBP-p40 I LBP-ps7) also dacraasad in TS calls. Northern Molllng anatysis revealed that the tmnscriptionafs of LBP-p40 was also inhibited h some TS patlsnls, while ll was not affacted in other paIients. Thus, in TS patlsnls. the expression of LBP-p40 wassuprassad silhar in transctiptbnal level or in translatIonal Ibvel.