정상림프구, 각화세포인 HaCat 세포 및 태아세포인 MRC-5 세포에서 DNA polymerase α를 특정하게 억제하며 DNA 복제 에 일차적으로 관여하는 물질로 알려진 aphidicolin을 처리하여 나타나는 fragile sites를 알기 위하여 각 세포군에 aphidicolin 0.05~0.15 μg/ml를 배양세포를 수확하기 24시간 전에 처리하여 각 세포군의 염색체를 검경한 결과 aphidicolin의 양은 0.15 μg/ml를 처리했을 때 fragile site가 나타나는 빈도가 최적이었으며 각 군마다 나타나는 빈도의 순서는 MRC-5 세포, 림프구 그리고 HaCat 세포의 순이었다. 각 군에서 공통적으로 높은 빈도로 fragile site가 나타나는 부위는 16q23였으며 태아세포에서 높은 빈도로 나타나는 부위는 1p31였다. 이 결과는 염색체 16q23에 있는 유전자 혹은 핵산은 aphidicolin에 매우 민감한 부위라는 것을 의미하며 발생중인 세포에서는 1p31 부위에 있는 유전자 혹은 핵산이 aphidicolin에 매우 민감한 부위라는 것을 의미하는 것으로 생각된다.
To investigate fragile sites induced by aphidicolin which is a specific inhibitor of eukaryotic DNA polymerase α which is primarily associated with chromosomal DNA replication in human lymphocytes, HaCat cells (human keratinocytes) and MRC-5 cells (human embryonic lung fibroblast), we cultured each cells in RPMI 1640 with 10% fetal calf serum and 2% PHA. Treatment of the cells with aphidicolin was generally carried out for the last 24 hours of culturing. The drug was dissolved in DMSO and used at final concentrations of 0.05~0.15 μg/ml, corresponding to a maximum DMSO concentration of 0.028%. Karyotypes of each cells were performed by routine method, and 50 metaphases were scored for each culture for analysis of breakage rate. Experimental cells treated with APC showed a dose dependent sensitivity and the amounts of chromosome breakage induced by APC are the highest in concentration of 0.15 μg/ml. The frequency of fragile sites on each cells appeared in MRC-5 cells, lymphocytes and HaCat cells in order. The common fragile sites on all experiments was 16q23, and the common fragile sites on embryonic cells was 1p31. It can be concluded that gene or nucleic acid which is located on 16q23 is the most important factor to induce chromosomal breakage with sensitivity to aphidicolin and 1p31 is important site to induce chromosomal breakage in embryonal cells.
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