In situ thermal denaturation of proteins in dunning AT-1 prostate cancer cells:: Implication for hyperthermic cell injury
In situ thermal denaturation of proteins in dunning AT-1 prostate cancer cells:: Implication for hyperthermic cell injury
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DOI：
10.1114/b:abme.0000042226.97347.de
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发表时间：
2004-10-01
影响因子：
3.8
通讯作者：
Bischof, JC
中科院分区：
文献类型：
--
作者：
He, XM;Wolkers, WF;Bischof, JC
本研究探讨了Dunning AT-1前列腺肿瘤细胞原位热蛋白变性及其与细胞直接热损伤的关系。采用傅里叶变换红外光谱（FTIR）和差示扫描量热法（DSC）研究了蛋白质的原位热变性。不同温度下的FTIR光谱显示了AT-1细胞内蛋白质原位热变性过程中蛋白质二级结构的变化（从螺旋到延伸的β折叠）。用DSC进行的量热研究表明，吸热热释放与原位热蛋白质变性有关。此外，无论是通过FTIR检测到的二级结构的变化和DSC检测到的量热变化进行了定量和动力学的整体原位热蛋白质变性在不同的加热条件下推导。发现最初可检测到整体原位热蛋白质变性的起始温度与扫描速率有关（类似于41 ℃时2 ℃ min（-1）和类似于44 ℃时5 ℃ min（-1））。整体原位热蛋白质变性的动力学来自DSC和FTIR测量，并使用动力学和统计模型拟合。FTIR和DSC在相同加热条件下测定的动力学数据吻合较好。整个原位热蛋白质变性的活化能被发现强烈地依赖于所考虑的温度范围（活化能范围从44和90 ℃之间的110 kJ mol（-1）到44和50 ℃之间的750 kJ mol（-1））。然而，它对加热速率的依赖性是可以忽略的。几个变性峰，包括一个在62和65 ℃之间的主要变性峰，从DSC和FTIR结果中可以识别。为了直接研究热诱导的细胞损伤和原位热蛋白变性之间的关系，使用在与DSC蛋白研究相同的条件下制备的AT-1细胞定量急性（碘化丙啶染料排除，热处理后3小时评估）和慢性（克隆形成，热处理后7天评估）细胞损伤。细胞损伤研究结果与DSC蛋白质变性研究结果的比较表明，整体原位热蛋白质变性与急性和慢性细胞损伤均具有良好的相关性，提示整体原位热蛋白质变性是AT-1细胞在大分子水平上直接热损伤的重要机制。
The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure ( from a helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (similar to41degreesC at 2degreesC min(-1) and similar to44degreesC at 5degreesC min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from similar to110 kJ mol(-1) between 44 and 90degreesC to similar to750 kJ mol(-1) between 44 and 50degreesC). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between similar to62 and 65degreesC, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at themacromolecular level.