２２回日本研究皮膚科学会総会・抄録

２２回日本研究皮膚科学会総会・抄録

-61- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
MUTATIONS OF THE INK4A LOCUS IN BASAL CELL CARCINOMAS.
Yoshiaki Kubo, Yoshio Urano, Seiji Arase.
Department of Dermatology, The University of Tokushima, School of Medicine, Tokushima, Japan.
The INK4a locus encodes two different proteins, p16INK4a and p19ARF. p16INK4a is an inhibitory protein of cyclin-dependent kinases (Cdks) 4 and 6, which regulate the cell cycle. p19ARF also has the ability to arrest cell proliferation at both G1 and G2 of the cell cycle. We previously detected mutations of the INK4a locus in 3 out of 21 squamous cell carcinomas of the skin (Kubo et al., BBRC in press). However, no information is available about mutations of the INK4a locus in basal cell carcinomas (BCCs). In this study, mutations in all coding regions in the INK4a locus (exons 1b, 1a, 2, and 3) were screened in 25 BCCs by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP). Extra bands showing altered migrations were detected in some of these BCCs. DNA sequence analysis showed a nonsense mutation of CGA→TGA at codon 80 of the p16INK4a gene in one BCC. DNA sequence analysis of other BCCs with extra bands is under investigation. We concluded that inactivation of the INK4a locus has some relevance to the carcinogenesis in at least some of BCCs.

-62- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
EXPRESSION OF HUMAN PATCHED GENE IN VARIOUS SKIN NEOPLASMS.
Tohru Nagano, Masato Ueda, Masamitsu Ichihashi.
Department of Dermatology, Kobe University School of Medicine, Kobe, Japan.
The gene for nevoid basal cell carcinoma syndrome ( PTCH ) has been identified and encodes a protein with strong homology to the Drosophila segment polarity gene, patched. Mutations of PTCH were also found in one-third of sporadic BCCs,which were accompanied with overexpression of the transcript of PTCH. To screen the involvement of PTCH alterations in cutaneous carcinogenesis,we analyzed the mRNA level of PTCH by RT-PCR method in various skin tumors by assuming that the overexpression of mRNA of PTCH is due to gene alteration. Analysis of 11 BCCs,4 SCCs, 2 Bowen's diseases, 2 keratoacanthomas , 3 actinic keratoses , 2 malignant melanomas and 3 normal skin showed that 9 of 11 BCCs expressed higher levels of mRNA expression of PTCH compared with other samples. These preliminary data may indicate that abnormalities of PTCH are rather specific for the development of BCC.

-63- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
RNASE SENSITIVE, CYTOPLASMIC SIGNALS BY IN SITU HYBRIDIZATION WITH HPV PROBES IN KERATOACANTHOMAS.
Hong Duan1, Shinichi Imafuku1, Jun Tsujita2, Shin-ichiro Yasumoto1.
1Department of Dermatology, Faculty of Medicine, Kyushu University, 2Department of Dermatology, School of Medicine, Fukuoka University, Fukuoka, Japan.
Human papillomavirus (HPV) has been detected in various epidermal and mucosal tumors including squamous cell carcinoma(SCC), Bowen's disease(BD) and keratoacanthoma(KA). We have examined the presence of HPV in KA by in situ hybridization with pan HPV probe. Formalin-fixed paraffin-embedded tissue sections were used for hybridization. Three out of eleven KA showed strong positive signals in the cytoplasm of tumor cells. These cytoplasmic signals were not altered by DNase digestion, but disappeared after RNase treatment. These cells were negative by polyclonal anti-HPV antibody staining. These signals were not detected in any SCC nor BD examined. These findings propose the idea of different mode of viral integration/replication in these tumors.

-64- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
EXPRESSION OF ONCOGENE C-FOS IN NEUROFIBROMA CELLS: MODULATION BY GROWTH FACTOR AND VITAMIN D3 ANALOGUE.
Juichiro. Nakayama, Kazunori. Urabe.
Department of Dermatology, Kyushu University Faculty of Medicine,Fukuoka, Japan.
Previously we reported that vitamin D3 analogues inhibited the growth of neurofibroma (abbreviated as NF) cell lines isolated from neurofibromas in patients with von Recklinghausen's neurofibromatosis-1. Also, we found that local injection of a vitamin D3 analogue, 22-oxacalcitriol (OCT), caused remarkable decrease in the density of the NF cells subcutaneously transplanted into nude mouse skin. As the next investigation, we analyzed the expression of oncogene, c-fos, in the NF cell lines by PCR methods for clarifying the mechanisms of the growth inhibition by OCT. We found that the NF cell lines expressed c-fos under the serum-depleted conditions, while normal fibroblasts did not. In addition, the expression of c-fos in the NF cell lines was augmented by such growth factors as bFGF and NGF. Concerning the effect of OCT on the c-fos expression, OCT rather augmented the expression within 15 min after addition of the vitamin D3 analogue in the NF cell lines. These results indicate that the expression of c-fos in the NF cell lines is constitutive and kinetics of the modutation by growth factors or OCT are quite different from those in normal fibroblasts.

-65- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
A NEW MODEL OF IN ViVO TUMOR ANGIOGENESIS ESTABLISHED BY MIXED CULTURE OF HUMAN TUMOR CELLS AND MURINE MICROVASCULAR ENDOTHELIAL CELL LINE, F-2.
Ken-Ichi Toda1, Naoko Kimura2, Reiji Kannagi2, Sadao Imamura1.
1Deptment of Dermatology Fac. Kyoto University, 2Dept. of Pathology Aichi Cancer Center, Aichi, Japan.
Although it is important to learn the interaction between tumor cells and microvascular endothelial cells(EC) for investigating the tumor angiogenesis, there have been no good animal models to substantiate it. In the present study, the intra-cutaneous innoculation system of the mixture of human tumor cells and F-2 in nude rats was established. The epidermoid cells,A431, colon cancer cells, Colo-201, and melanoma cells, Mevo were used as human tumor cells. The cutaneous tumor induced by the mixed culture of A431 or Mevo and F-2 in nude rats was 3-5 times larger than that by tumor cells alone. The size of the induced tumor by the mixture of Colo 201 and F-2 was not significantly increased. The induced tumor by the mixed cultures were composed of the tumor nests and F-2-derived functioning tumor vessels filed with host blood cells, but the induced tumor by human tumor cells alone revealed central necrosis of tumor mass lacking the tumor angiogenesis. The tumor formation by the mixed culture were markedly suppressed by the anti-sialyl Lex, Lea, andr integrin antibodies. These observations indicate that the new tumor-bearing animal model established by the mixture of some human tumor cells and F-2 is useful for the study of tumor cell-EC interactions and in vivo tumor angiogenesis.

-66- Carcinogenesis/Tomor/Oncogene(1) 9/4 Room-D
ANTI-TUMOR ACTIVITY OF IL-12 AGAINST ANGIOSARCOMA.
Mikio Masuzawa, Naomichi Hara, Yuko Hamada, Takao Fujimura, Shigeo Nishiyama, Kensei Katsuoka.
Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Japan.
IL-12 called as NK cell stimulatory factor is well known to have potent anti-tumor activity in a number of murine tumor models. In the present study we examined anti-tumor activity of IL-12 in a BALB/c mouse model of NK-sensitive murine angiosarcoma(AS) ISOB-1 that we have established. Subcutaneous ISOB-1 tumors were treated with IL-12 (0.1microgram x 10 times in 2wk) injected intra-lesionally. This local immunotherapy demonstrated complete rejection of the tumors. Additionally the rejected mice inhibited existence of ISOB-1 cells, but not Meth-A cells re-injected intraperitoneally. The infiltrating cells into peritoneal cavity showed in vitro cytotoxic activity against ISOB-1 and NK-sentive YAC-1 cells, but not Meth-A cells. The activity was not influenced by depletion of CD4- or CD8-positive cells from them. Therefore, the intra-lesional injection of IL-12 showed potent anti-tumor activity against AS with induction of systemic AS-specific protective immunity by NK cells but not CTL. Our results could suggest that intra-lesional IL-12 injection will be an optimum treatment for fatal AS arising on the scalp.