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

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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.