本研究為探討 paclitaxel 對蛋白質磷酸化的影響，我們先以本實驗室熟悉的大鼠腦瘤細胞 9L 為材料，以 10-6 M paclitaxel 處理 6 小時後，以 [32P] orthophosphate 標幟兩小時後發現 57kD 蛋白質磷酸化程度大幅提高，我們以岡井田酸 (okadaic acid) 及 熱休克 (heat shock) 處理，配合西方氏墨點(Western blot)實驗，結果可確認此 57kD 蛋白質為 vimentin。此 paclitaxel 對 vimentin 過度磷酸化的作用有濃度及時間的依存性，以 10-6 M 及 12 小時最好，為瞭解此種活性由何種蛋白質激脢所作用，我們則用一系列激脢抑制劑處理，結果顯示 10 nM的 starosporine 及 200 nM 的 BIM 可抑制vimentin此種磷酸化，顯示此過程可能由 protein kinase C 作用。在細胞形態及細胞骨架重組的探討方面，我們先以掃描式電子顯微鏡觀察，發現 pacliatxel 的處理會使正常生長的 9L 細胞由平貼的形態逐漸圓起 (round up)，由於細胞形態由細胞骨架支撐，所以我們以再以螢光顯微鏡觀察細胞骨架的組成，發現 vimentin 中間絲蛋白幾乎瓦解，並堆積在細胞核附近，此結果證明 paclitaxel 誘發 vimentin 的磷酸化現象是伴隨著結構的重組及整個細胞形態的變化。
有上述成果後，我們以人類非小細胞肺癌細胞株 H460 及其抗 paclitaxel 的抗藥性亞株 H460/TAX 為對象，發現 paclitaxel 除了誘發 vimentin 的過度磷酸化之外，亦造成 40 kD 的蛋白質的過`度磷酸化，以 Western blot 實驗證明此 40 kD 的磷酸蛋白為另一種中間絲蛋白即角質蛋白-19 (keratin 19, K19)，在細胞骨架的組織變化上，H460與 H460/TAX 的 K19 亦類似 9L 的 vimentin ，在 paclitaxel 的作用下伴隨過度磷酸化而逐漸瓦解，並向細胞核堆積。
在濃度與時間效應的探討中我們發現 H460/TAX 比 H460 需要更高的藥物濃度與更長的作用時間方能使 K19達到最高的磷酸化程度，此差異可能由於細胞攝取藥物的量不同或其它活性差異導致，因此我們再以碳-十四標定的 7-([carbonyl-14C]-acetyl)paclitaxel 為追蹤劑來測定兩者的藥物累積量，結果顯示 H460/TAX 單位時間的藥物累積量少於 H460。但H460/TAX 的 P-glycoprotein 與 MRP 都沒有過度表現，顯示尚有未知的運輸蛋白主導著 H460/TAX 的細胞藥物攝取量。 K19 中間絲蛋白磷酸化與非小細胞肺癌抗藥性的關係是值得我們日後再進一步探討。

we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at10-6 M of taxol treatment for 8 h and diminished at higher (10-5 M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10-6 M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxoltreated cells, whereas the microtubules and the actin microfilaments were less affected. Based on these finding, we further study the mechanism of paclitaxel resistance in paclitaxel resistance. We have established a paclitaxel-resistant mutant cell line called H460/TAX, which was derived from human non-small cell lung cancers (NSCLC) H460. A 64-fold more resistant was shown in our assay compared with the parental cells. High specificity of drug resistance was also observed in that this mutant was not cross-resistant to several other anticancer drugs. Drug acccumulation in H460/TAX was significantly less than that in H460. Many endogenous protein profiles were intact, including the expression level of P-glycoprotein, multidrug resistance-associated protein, the 70 kDa heat shock proteins as well as the phosphorylation of Bcl-2 in H460/TAX cells except that total amount of a- and b-tubulins was higher in H460/TAX than in H460 cells. Higher drug concentration and longer treatment for paclitaxel were required in H460/TAX to exert the phosphorylation of keratin 19 which was then accompanied by reorganization of the intermediate filament and the microtubule networks. The fact that H460/TAX defects only in paclitaxel-targeted pathway and leaving intact with those previous proposed factors suggesting a yet identified factor might be independently involved in the mechanism of paclitaxel resistance in human lung cancers.

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