摘 要 流行病學的研究顯示砷化物是人類致癌劑，但其致癌機轉尚待查明。在本實驗室黃錫慶博士的研究指出HeLa S3細胞經過5 μM亞砷酸鈉處理，會有大約35%的細胞停滯在有絲分裂中期，而處理staurosporine (SSP,蛋白質激酶抑制劑)之後，會促使中期停滯細胞離開有絲分裂期。本研究的目的為探討SSP如何讓亞砷酸鈉誘引的中期停滯細胞離開有絲分裂期。藉由免疫螢光染色法觀察有絲分裂期之細胞次級結構：紡錘體及DNA的構形，使用西方點墨法來偵測細胞中Pds1的含量。結果顯示亞砷酸鈉誘引的中期停滯細胞經過50 nM SSP的處理後，會促使Pds1的降解，同時也伴隨著細胞質分裂。除此之外，藉由二維電泳的分析，觀察到亞砷酸鈉誘引的中期停滯細胞，經過SSP的處理後，其蛋白質表現的改變。藉由MALDI-TOF來鑑定這些蛋白質的種類。在SSP的處理下，觀察到carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein等蛋白質的改變。另外，將nocodazole及亞砷酸鈉誘引的中期停滯細胞的紡錘體，藉由二維電泳法分析比較兩種處理下ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein等之蛋白質發生改變。這些參與有絲分裂停滯機制之蛋白質值得進一步的探討。 Abstract Arsenic is a well-documented human carcinogen, primarily based on the evidence of clinical observation and epidemiological studies. Previous studies in our laboratory have shown that treatment of HeLa S3 cells with 5 μM arsenite leads to approximately 35% of the cells arrested in the mitotic metaphase. Treatment of arsenite-arrested mitotic metaphase cells with staurosporine (SSP, a protein kinase inhibitor) enforces them exiting from mitosis. In this thesis, experiments were conducted to ask how SSP enforces the mitotic exit. The microscopic structure of mitotic spindles and chromosomes were examined by immunofluorescent technique using anti-β-tubulin antibody and DAPI, and the content of Pds1 were analyzed by Western blot technique. The results show that treatment of arsenite-arrested mitotic cells with 50 nM SSP leads to degradation of Pds1 and increases in the frequency of cytokinesis. The proteins modified by SSP on arsenite arrested mitotic cells are analyzed by two-dimensional electrophoresis. These proteins are identified by MALDI-TOF technology. Several proteins are apparently modified by staurosporine treatment, for instance, carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein etc. Two-dimensional electrophoresis also detects differentially expressed proteins between the nocodazole and arsenite-arrested mitotic spindle fractions. These proteins are ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein. The involvement of these proteins in mitotic arrest warrants further investigation.
