Use of Fluorescence In Situ Hybridization Technique to Predict the Radiosensitivity of Fibroblasts and Nasopharyngeal Cancer Cells

• Main Author: 劉淑貞
• Other Authors: Fu-Du Chen
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/09090608068012384231

碩士 === 國立陽明大學 === 放射醫學科學研究所 === 88 === Abstract A wide variety of assays are currently being explored for predicting either the intrinsic radiosensitivity of individual tumors or their response to radiotherapy. The most important goal of a predictive assay is to obtain information that can be used to choose a suitable treatment protocol, so that each individual patient can achieve better treatment result. There are assay systems being studied and applied to the clinic. These include: 1). cell surviving fraction at 2 Gy (SF2), 2). growth assays, e.g. MTT assay, 3). The measurement of radiation-induced DNA damage, e.g. comet assay, 4).The measurement of chromosomal damage, such as FISH and premature chromosome condensation. 5). The measurement of tumor hypoxia, including the use of oxygen electrodes, the detection of fluorine-labelled misonidazole using PET. 6). Tumor cell repopulation, e.g. measure the potential doubling time (Tpot) of tumor etc . In the present studies, the Fluorescence in situ hybridization (FISH) using whole chromosome probes for chromosome 1,2,4 (labeled with Cy3) and 3,5,6 (labeled with FITC) was used to investigate the radiosensitivity of NPC cells and fibroblasts. Spectral Karyotyping technique (SKY) and comparative genomic hybridization (CGH) techniques were also tested as potential substitutes for the prediction of radiosensitivity. The clonogenic assay was done as a standard reference. Six human NPC cell lines were tested. Cell surviving fractions were determined by clonogenic assays and correlated with chromosome translocation frequencies as detected by FISH technique after irradiation. Five fibroblasts cultured from different NPC patients were also tested by FISH. In general, for the radiosensitive cell lines, translocation frequencies were increased after a given radiation dose. The level of chromosome translocation frequencies induced by radiation in vitro correlates well with cell survival as determined by clonogenic assays. When the given dose was split into two fractions, the level of cell survival became lower for all six cell lines. This may possibly due to the cell cycle reassortment effect after irradiation. The use of SKY technique revealed a full genomic translocation pattern, which is totally different from the results calculated by using the Lucas equation. This suggests that the Lucas equation may be not suitable for measuring translocation frequencies in aneuploid tumor cells. The DNA content of cells seems to play an important role in determining in vitro radiosensitivity. In general, increasing DNA ploidy can increase cell surviving fraction. The spontaneous translocation frequencies of fibroblasts from NPC patients were much higher than that observed in normal human lymphocytes, this may be related to a). the fibroblasts have longer life-span. B). the NPC patients might have exposed themselves to a poor environment for a longer period of time. These factors lead to a larger accumulation of stable chromosome aberrations. There was no significant difference observed with and without 3.6Gy irradiation as compared with intrinsic DNA copy number changes. Among the six NPC cell lines, the deletion of chromosome 3p and the amplification of chromosome 20q were seen in all cells. Gains of DNA sequences were more frequent than loss. Some of the most frequent DNA copy number changes were found both in NPC cell lines and primary NPC tumors, this may indicate the possible involvement of some oncogenes , tumor supressor genes or DNA repair genes on these spots in the carcinogenesis of NPC. The DNA copy number changes were also found in chromosome 4 and 7 of fibroblasts from NPC patient. From these results, the use of FISH technique to measure chromosome translocation frequencies is an ideal substitute for conventional clonogenic assays. However, the major difficulty at present studies is to obtain sufficient numbers of metaphases from small tumor biopsies within a short period of time. Although CGH is a powerful genome-wide screening method, but was found not suitable for the sensitivity prediction . Finally, the SKY technique may be a potential method for future radiosensitivity prediction for its accuracy, reliability and information provided in it.