Evans D P, Tomasovic S P
Department of Tumor Biology, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston 77030.
Int J Hyperthermia. 1989 Sep-Oct;5(5):563-78. doi: 10.3109/02656738909140481.
The role of calmodulin (CaM) in hyperthermic cell killing, and the development of thermotolerance in rat 13762NF mammary adenocarcinoma cells, was investigated by using the CaM antagonists W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and W-13 [N-(4-aminobutyl)-2-chloro-naphthalenesulphonamide] and their less active analogues W-5 [N-(6-aminohexyl)-1-naphthalenesulphonamide] and W-12 [N-(4-aminobutyl)-2-naphthalenesulphonamide]. The CaM antagonists W-7 and W-13 potentiated 43 degrees C cell killing (and the less active analogues did not) at a concentration compatible with CaM inhibition, thus hyperthermic perturbation of CaM-regulated processes may contribute to cellular lethality. The potentiation of hyperthermic killing by antagonists appeared to be temperature-dependent, sensitizing much more effectively to 43 degrees C than to 42 degrees C killing. The effect may be related to differing primary mechanisms of hyperthermic killing activated at the two temperatures, or simply to differences in incorporation or localization of the antagonists. The presence of the CaM antagonists throughout fractionated 42 degrees C or 43 degrees C heating, or during continuous 42 degrees C heating, did not significantly inhibit or potentiate the triggering and development of thermotolerance or alter the rates of heat stress protein (HSP) synthesis. Studies using CaM-agarose isolation of CaM-binding proteins indicated that binding of some HSP to CaM-agarose occurred and was Ca2+-dependent. The specificity and physiological relevance of these HSP binding to CaM was not clear, since their affinity was not high in these cells. Presumably W-7 would perturb any physiologically relevant CaM-protein interactions in cells but W-7 concentrations that reduced HSP and other protein binding to CaM-agarose columns by 50 per cent or more, had no effect on thermotolerance development in cells. These observations, combined with the studies that showed little effect of CaM antagonists on HSP synthesis at concentrations which potentiated cell killing, suggested that events leading to triggering or developing thermotolerance were not strongly dependent on any putative HSP binding to CaM. These studies also suggest some targets of hyperthermic cell killing at 43 degrees C are different from those that lead to the triggering and development of thermotolerance.
通过使用钙调蛋白拮抗剂W - 7 [N -（6 - 氨基己基）- 5 - 氯 - 1 - 萘磺酰胺]和W - 13 [N -（4 - 氨基丁基）- 2 - 氯 - 萘磺酰胺]及其活性较低的类似物W - 5 [N -（6 - 氨基己基）- 1 - 萘磺酰胺]和W - 12 [N -（4 - 氨基丁基）- 2 - 萘磺酰胺]，研究了钙调蛋白（CaM）在大鼠13762NF乳腺腺癌细胞热诱导细胞杀伤及热耐受形成中的作用。钙调蛋白拮抗剂W - 7和W - 13在与钙调蛋白抑制作用相适应的浓度下增强了43℃时的细胞杀伤作用（而活性较低的类似物则没有），因此钙调蛋白调节过程的热扰动可能导致细胞死亡。拮抗剂对热诱导杀伤的增强作用似乎具有温度依赖性，对43℃杀伤的致敏作用比对42℃杀伤更为有效。这种效应可能与在这两个温度下激活的热诱导杀伤的不同主要机制有关，或者仅仅与拮抗剂的掺入或定位差异有关。在42℃或43℃分级加热全过程中，或在42℃持续加热过程中存在钙调蛋白拮抗剂，均未显著抑制或增强热耐受的触发和形成，也未改变热应激蛋白（HSP）的合成速率。使用CaM - 琼脂糖分离钙调蛋白结合蛋白的研究表明，一些HSP与CaM - 琼脂糖发生结合，且这种结合依赖于Ca2 +。这些HSP与钙调蛋白结合的特异性和生理相关性尚不清楚，因为它们在这些细胞中的亲和力不高。推测W - 7会干扰细胞中任何生理相关的钙调蛋白 - 蛋白质相互作用，但能使HSP和其他蛋白质与CaM - 琼脂糖柱的结合减少50%或更多的W - 7浓度，对细胞热耐受的形成没有影响。这些观察结果，结合那些表明钙调蛋白拮抗剂在增强细胞杀伤作用的浓度下对HSP合成影响很小的研究，表明导致热耐受触发或形成的事件并不强烈依赖于任何假定的HSP与钙调蛋白的结合。这些研究还表明，43℃时热诱导细胞杀伤的一些靶点与导致热耐受触发和形成的靶点不同。
