中文摘要
六價鉻化物是廣泛存在的環境物質，已被評估為人類致癌物，六價鉻之基因毒性及致癌性與它在細胞內被還原活化成五價、四價及三價鉻之過程產生的自由基有高度關係。細胞週期運轉之失控是癌形成之一重要因素。然而，六價鉻影響細胞週期運轉的機制仍不十分清楚。本篇論文探討六價鉻干擾人類肺癌細胞CL3之細胞週期進行，以及鉻對核酸複製之前或後期細胞的基因毒性。六價鉻會導致未同步化的細胞延滯在S期。由逆流離心細胞分離系統所分離出的G1期細胞，在六價鉻的處理之下，也同樣產生S期延滯，且六價鉻並未造成明顯的G1延滯。利用Aphidicolin將細胞同步化在細胞週期G1/S臨界期與early G2期，處理20 mM六價鉻兩小時後繼續培養細胞2-10小時，以流式細胞儀分析發現細胞延滯在G2/M期。再者，以倒立式顯微鏡計算有絲分裂期的細胞數，確認六價鉻延緩細胞進入M期。進而以西方墨點法及免疫沈澱激酶活性分析法來檢測調控細胞週期進行之分子，發現六價鉻使cdc2激酶活性延遲兩小時才被活化，會減緩cyclin B1的分解，且使活化後之cdc2激酶也延遲四小時才失去活性。因此推測六價鉻應是延緩G2進入M期以及跳脫M期。六價鉻對同步化在G1/S臨界期或early G2期細胞造成的細胞毒性無明顯差異。此外，六價鉻對G1/S臨界期或early G2期細胞都會誘發高於控制組約兩到三倍的微核生成。前處理維生素C會加強六價鉻處理之G1/S細胞延滯在G2/M期，加強延緩cyclin B1之分解，但不影響鉻干擾cdc2激酶活性，故推測維生素C會加強六價鉻延遲脫離M期的現象。前處理維生素C只些微增加控制組及六價鉻誘發的微核數目。綜合以上，六價鉻會造成細胞週期延滯在S、G2與M期，但卻沒有明顯誘發細胞停滯在G1期。六價鉻不太誘引G1暫停，可能導致受損傷的核酸經過不正常的複製，因而誘使基因缺失及癌形成。

Abstract
Cr(VI) compounds are ubiquitous environmental toxicants that have been evaluated as human carcinogens. Cr(VI) genotoxicity and carcinogenicity are highly associated with the generation of reactive species including Cr(V), Cr(IV), Cr(III) and reactive oxygen species during its intracellular reduction. De-regulation of cell cycle progression is an important issue leading to cancer formation. However, the mechanism that Cr(VI) interferes with cell cycle progression is not fully understood. In this thesis, we have investigated the effects of Cr(VI) on cell cycle checkpoint control and genotoxicities of Cr(VI)-treated cells synchronized before and after DNA replication using human non-small lung carcinoma CL3 cells. In asynchronous cells, Cr(VI) exposure caused the delay of cell cycle at S phase. The Cr(VI)-elicited S phase delay was also observed in G1 phase cells collected from counterflow centrifugal cell elutriation system. Cr(VI) did not cause significant G1 delay. Cells were synchronized at G1/S border or early G2 phase using aphidicolin, exposed to 20 mM of Cr(VI) for 2 h, kept culture for 2-10 h and subjected for flow cytometry analysis. A significant G2/M delay were observed in both Cr(VI)-treated G1/S or early G2 cells. Mitotic figure analysis using an inverted microscope confirmed that Cr(VI) delays M phase entry. Using western blotting and immunocomplex kinase activity analysis techniques, we have observed that the activation of cdc2 kinase is delayed for 2 h, and the degradation of cyclin B1 and deactivation of cdc2 kinase are also delayed for 4 h. The results suggest that Cr(VI) delays G2 enter into M phase and the exit of M phase. Cr(VI) induced similar levels of cytotoxicity and genotoxicity in G1/S and early G2 cells. Ascorbic acid pretreatment could enhance the effect of Cr(VI) on the delay of G2/M phase and degradation of cyclin B1 but not the activity of cdc2 kinase in Cr(VI)-treated G1/S cells, suggesting ascorbic acid enhances the delay of M phase exit induced by Cr(VI). Ascorbic acid alone slightly increased micronucleus formation in both untreated and Cr(VI)-treated cells. Together, Cr(VI) causes cell cycle delay at S, G2 and M phases, however, it does not induce G1 arrest, which can allow abnormal replication of damaged-DNA and subsequently leading to gene deletion and carcinogenesis.

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