INVESTIGATION OF THE TUMOR SUPPRESSOR GENES INVOLVED IN THE CARCINOGENESIS OF ORAL SQUAMOUS CELL CARCINOMA BASED ON THE GENETIC COMPLEMENT.

1)含有来自正常人3号染色体的亚染色体可转移片段（STF）的A9细胞的构建。用2-12戈伊的X射线照射含有用pSV 2neo标记的正常人3号染色体的A9（neo 3）细胞。将辐照的微细胞与A9细胞融合，并在补充有10%CS和0.8mg/ml G418的DMEM中选择G418抗性A9（STF）克隆。我们构建了16个含有正常人3号染色体部分片段的A9（STF）克隆。为了鉴定含有STFs的A9克隆中的人类序列，我们用人类3号染色体特异性探针进行了Q-显带分析和染色体涂染。用3.2号染色体上的54个微卫星标记对16个A9（STF）克隆的DNA样品进行定位。2）亲本细胞系和A9（STF）克隆的致瘤性。亲代A9细胞皮下接种裸鼠后3 ~ 5周内，在裸鼠体内所有部位均形成进行性生长的肿瘤。另一方面，只有一个微细胞杂交体; A9（neo 3B）细胞在皮下注射后甚至超过6个月也未能形成肿瘤。另外5个A9（STF）克隆在裸鼠体内产生肿瘤，与亲本A9细胞相似。这些数据表明，在人3号染色体上可能与裸鼠肿瘤发生有关的关键抑癌基因仅在A9（neo 3B）细胞中表达。3）用抑制性消减杂交（SSH）鉴定人3号染色体上的关键抑癌基因。我们现在试图鉴定在抑制克隆中表达的关键抑癌基因，而不是在未抑制的克隆中，在人3号染色体上使用SSH方法。

1)Construction of A9 cells containing subchromosomal transferable fragments(STFs)derived from the normal human chromosome 3.A9(neo3)cells, containing normal human chromosome 3 tagged with pSV2neo, were irradiated with doses of 2-12 Gy of X-rays. Irradiated microcells were fused to A9 cells, and G418-resistant A9(STF)clones were selected in DMEM supplemented with 10% CS and 0.8mg/ml of G418. We have constructed 16 A9(STF)clones containing partial fragment derived from normal human chromosome 3. To identify the human sequence in the A9 clones containing STFs, we performed Q-banding analysis and chromosome painting with the human chromosome 3 specific probe. To localize the isolated STFs, DNA samples prepared from all 16 A9(STF)clones were assayed using 54 microsatellite markers on chromosome 3.2)Tumorigenicity of the parental cell line and A9(STF)clones.The tumorigenicity of the parental A9 cells and 6 different A9(STF)clones was examined in nude mice. The parental A9 cells formed progressively growing tumor in all the sites of nude mice within 3 to 5 weeks after subcutaneous inoculation. On the other hand, only one microcell hybrid ; A9(neo3B)cells failed to form tumors even more than 6 month after subcutaneous injection. Another five A9(STF)clones produced tumor in nude mice like the parental A9 cells. These data suggested that the critical tumor suppressor gene(s)which may contribute to the development of tumor in nude mice on human chromosome 3 was expressed only in the A9(neo3B) cells.3)Identification of the critical tumor suppressor gene(s)on human chromosome 3 using suppression subtractive hybridization(SSH).We are now trying to identify the critical tumor suppressor gene(s), which was expressed in the suppressed clone, not in the unsuppressed clones, on human chromosome 3 using SSH method.