023-C1 Tumor biology 4. The Biological Effect of All-trans Retinoic Acid on Human Oral Cancer Cells
Kao, S. y.1, Wong, D. T. t~. l Oral and Maxillofacial Surgery, Department of Dentistry, Veterans General Hospital-Taipei School of Dentistry, National Yang-Ming University, Taiwan, R.O.C., 20ral Pathology, Harvard School of Dental Medicine, USA Retinoids have been reported to retard the growth of cancer. However, it is still not clear whether tumor suppression is the result of inducing differentiation, inhibiting proliferation or some other mechanism. In our pilot study, we utilized two human oral cancer cell lines (SCC15, SCC25), harvested from tongue and floor of mouth, to demonstrate the retinoic acid(RA)-induced cell growth inhibition in the presence of RA at 1 gM. To explore the potential mechanism of RAinduced suppressed phenotype of human oral cancers, we used RT/PCR based m R N A differential display to screen the RA-inducible/suppressible genes. The amplified gene expression signals of RNA isolated from normal keratinocytes, human oral cancer cells and RA-treated human oral cancer cells were compared side by side. The candidate genes were isolated, purified and further verified by RT/PCR using gene specific primers. The initial result showed that 3 RA-inducible candidate genes, G15, T6, and T9, were found. The Genomic Southern blot analysis for RFLP probed with T9 showed an interesting pattern in SCC25, which is different from other clones.
5. High Induction of Poly(ADP-ribose) Polymerase in a Radioresistant KB Cell Line
Yanagisawa, T.1, Yamamoto, y2, Takahashi, y1, Urade, M.' 1Department of Dentistry and Oral Surgery, 2Department of Genetics, Hyogo College of Medicine, Hyogo, Japan It has been considered that the acquisition of radioresistance in mammalian cells is closely related to the repair of D N A damage; however, the radioresistance mechanism of human tumor cells is still unclear. Recently, it has been reported that poly(ADP-ribose) polymerase (PARP)-deficient cell lines are highly sensitive to UV- and X-irradiation and a variety of antitumor agents as compared to parental cell lines. Therefore, we investigated the relation between gene expression of PARP and radioresistance in a human K B carcinoma cell line. In this study, we used the cell line N10, which was isolated by treatment with 5mg/ml of bleomycin and 10 Gy of X-irradiation and was 3.1-fold more resistant to X-irradiation than the parental KB cell line. To examine PARP gene expression in N10, Northern blot analysis was performed by using a 32P-labeled PARP cDNA probe. The level of PARP m R N A was higher in N10 than in the KB cell line under the unirradiated condition. When these cells were irradiated at 4 Gy, the PARP m R N A level in KB decreased in a time-dependent manner, whereas that
in N10 remained high as much as 6 h after irradiation. An assay for PARP activity demonstrated an approximately 3.0fold higher activity in N10 than in KB under the unirradiated condition. X-irradiation caused a rapid induction of PARP activity within 1 h in both cell lines, but pretreatment of cells with nicotinamide, a PARP inhibitor, inhibited the enzyme induction and potentiated the radiosensitivity in N10, but not in KB. These results indicate that the expression of PARP is strongly involved in the radioresistance of the N10 cell line.
6. Accumulation of Oligoclonal T Cells in Oral Squamous Cell Carcinoma
Mouri, T., Nakamura, S., Sasaki, M., Shinohara, M., Ohyama, Y, Hiroki-lkebe, A., Kadena, T., lkebe, T., Tsunawaki, S., Shirasuna, K. Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, Fukuoka, Japan Oral squamous cell carcinomas (OSCC) are often infiltrated by a large number of T lymphocytes. To clarify the nature of the tumor-infiltrating lymphocytes (TIL) in OSCC, we first examined the T cell receptor (TCR) Vc~ and VJ3 gene usage by TIL and peripheral blood mononuclear cells (PBMC) obtained from 10 patients, using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In contrast to PBMC, TIL showed more restricted usage of V~ gene families while two unique VI3 gene transcripts (VI3 5.2 and VI3 6) were overexpressed in the TIL of more than half of the patients. We further sequenced PCR products from the overexpressed gene transcripts in TIL and the frequent usage of V[3 6/Jb 1.1 and VJ3 6/Jb 2.7 combinations by the TIL was observed. These results suggest that unique T cell subpopulations in TIL are clonally expanded. To confirm the oligoclonality of TIL, we examined the clonotypes of TCRI3chain gene transcripts expressed in TIL, regional lymph nodes (LN), and PBMC by RT-PCR and subsequent single strand conformation polymorphism (SSCP) analysis. This analysis revealed that the number of distinct bands in TIL or regional LN was greater than that in PBMC. Furthermore, distinct bands migrating to the same position on the gel were frequently detected in samples obtained from different locations in a patient. These results suggest that oligoclonal T cell populations containing identical T cell clonotypes accumulate in different regions of a patient. This study thus supports the existence of antigen-driven immune responses to OSCC in vivo.