Stevens J, Stevens Y W
Cancer Res. 1979 Oct;39(10):4011-21.
The physicochemical properties of nuclear and cytosolic glucocorticoid-binding components from corticoid-sensitive (CS) and corticoid-resistant (CR) mouse lymphoma P1798 cells have been compared. Nuclei or cytosol fractions were prepared from lymphocytes that had been labeled at 37 or 4 degrees, respectively, with 30 nM [3H]triamcinolone acetonide ([3H]TA). [3H]TA was extracted with 0.6 M KCl, 10 mM spermidine, or 4.5 mM MgCl2 from CS nuclei and with 0.6 M KCl or 10 mM spermidine from CR nuclei. As reported previously, nuclear-associated [3H]TA in CR cells was resistant to extraction with mM concentrations of MgCl2. Loss of bound steroid during extraction with 0.6 M KCl was minimized by including the chymotrypsin inhibitor, carbobenzoxy-L-phenylalanine, in the extraction buffer. The inhibitor was not required during extraction with spermidine or MgCl2. Nuclear and cytosolic extracts were examined by analytical agarose gel filtration and glycerol density gradient centrifugation under high salt (0.6 M KCl) conditions. The glucocorticoid-binding component in KCl, spermidine, and MgCl2 extracts from CS nuclei was considerably larger and more asymmetrical [Stokes radius, 57 to 59 A; sedimentation coefficient, 3.64 to 3.70S; molecular weight, 90,000 daltons; frictional ratio, 1.8; axial ratio (prolate ellipsoid), 15] than the [3H]TA-macromolecular complex in KCl and spermidine extracts from CR nuclei[Stokes radius, 29 A; sedimentation coefficient, 3.23 to 3.30S; molecular weight, 40,000 daltons; frictional ratio, 1.25; axial ratio (prolate ellipsoid), 5]. Control experiments showed that the smaller size of the glucocorticoid-binding component in CR nuclei was probably not due to cleavage of a larger, CS-like complex during the extraction procedure. The larger size of the CS [3H]TA complex did not appear to result from aggregation of s a smaller species. No difference in physicochemical parameters of the binding component was observed if cells were labeled with [3H]dexamethasone instead of [3H]TA. However, [3H]dexamethasone complexes were less stable than those formed with [3H]TA as indicated by considerable dissociation of [3H]dexamethasone during gel filtration and gradient centrifugation. This may be due to the 3- to 5-fold lower relative binding affinity of [3H]dexamethasone. Analysis of [3H]TA-labeled cytosol by gel filtration and gradient centrifugation revealed the presence of a single binding component with physicochemical properties similar to those of nuclear [3H]TA complexes from the same strain of tumor. These results suggest that previously described differences in extractability of nuclear-associated [3H]TA between the CS and CR strains of mouse lymphoma P1798 and the lack of response of CR P1798 to glucocorticoid administration may be due, at least in part, to the presence of an altered glucocorticoid-binding component in the resistant tumor cells.
对皮质激素敏感（CS）和皮质激素抵抗（CR）的小鼠淋巴瘤P1798细胞中核和胞质糖皮质激素结合成分的物理化学性质进行了比较。分别从在37℃或4℃下用30 nM [³H]曲安西龙丙酮化物（[³H]TA）标记的淋巴细胞中制备核或胞质部分。[³H]TA用0.6 M KCl、10 mM亚精胺或4.5 mM MgCl₂从CS核中提取，用0.6 M KCl或10 mM亚精胺从CR核中提取。如先前报道，CR细胞中与核相关的[³H]TA对毫摩尔浓度的MgCl₂提取具有抗性。在用0.6 M KCl提取过程中，通过在提取缓冲液中加入糜蛋白酶抑制剂苄氧羰基-L-苯丙氨酸，使结合类固醇的损失最小化。在用亚精胺或MgCl₂提取过程中不需要该抑制剂。在高盐（0.6 M KCl）条件下，通过分析琼脂糖凝胶过滤和甘油密度梯度离心法检查核提取物和胞质提取物。CS核的KCl、亚精胺和MgCl₂提取物中的糖皮质激素结合成分比CR核的KCl和亚精胺提取物中的[³H]TA-大分子复合物大得多且更不对称[斯托克斯半径，57至59 Å；沉降系数，3.64至3.70 S；分子量，90,000道尔顿；摩擦比，1.8；轴比（长椭球体），15] [斯托克斯半径，29 Å；沉降系数，3.23至3.30 S；分子量，40,000道尔顿；摩擦比，1.25；轴比（长椭球体），5]。对照实验表明，CR核中糖皮质激素结合成分较小的尺寸可能不是由于在提取过程中较大的、类似CS的复合物被裂解。CS [³H]TA复合物较大的尺寸似乎不是由较小物种的聚集导致的。如果用[³H]地塞米松而不是[³H]TA标记细胞，则未观察到结合成分的物理化学参数有差异。然而，[³H]地塞米松复合物比用[³H]TA形成的复合物稳定性更低，这在凝胶过滤和梯度离心过程中[³H]地塞米松的大量解离中得到体现。这可能是由于[³H]地塞米松的相对结合亲和力低3至5倍。通过凝胶过滤和梯度离心法对[³H]TA标记的胞质进行分析，发现存在一种单一的结合成分，其物理化学性质与来自同一肿瘤株的核[³H]TA复合物相似。这些结果表明，先前描述的小鼠淋巴瘤P1798的CS和CR株之间核相关[³H]TA可提取性的差异以及CR P1798对糖皮质激素给药缺乏反应可能至少部分归因于抗性肿瘤细胞中存在改变的糖皮质激素结合成分。
