Gilbert M, Knox S
Department of Radiation Oncology, Stanford University School of Medicine, California 94305-5105, USA.
J Cell Physiol. 1997 Jun;171(3):299-304. doi: 10.1002/(SICI)1097-4652(199706)171:3<299::AID-JCP8>3.0.CO;2-J.
Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na+/ K(+)-ATPase pump activity, as measured by the uptake of Rubidium-86 (86Rb+), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses > or = 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 microM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na+/ K(+)-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). 86Rb+ uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of 86Rb+ uptake, suggesting that the effects of radiation on the Na+/K(+)-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na+/K(+)-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD.
在转染的PW细胞中，Bcl-2过表达与辐射诱导的程序性细胞死亡（PCD）的抑制相关。我们之前报道过，在这些细胞中，辐射诱导的PCD抑制与膜超极化之间存在关联。在本文中，我们报道，通过铷-86（86Rb+）摄取量测定的Na+/K(+)-ATP酶泵活性，在Bcl-2过表达的PW细胞中显著高于对照PW细胞，并且在接受≥500 cGy剂量照射后，Bcl-2转染细胞中的泵活性降低程度小于对照细胞。当用能显著降低泵活性但不诱导PCD的哇巴因剂量（1 microM）孵育PW-Bcl-2细胞时，先前报道的与Bcl-2过表达相关的对辐射诱导PCD的保护作用不再存在。为了证明活性氧（ROS）影响Na+/K(+)-ATP酶泵活性，在照射前用N-乙酰半胱氨酸（NAC）孵育细胞，或用产生活性氧的药物丁硫氨酸亚砜胺（BSO）处理细胞。与未用NAC孵育而照射的细胞相比，用NAC孵育的照射细胞中86Rb+摄取量显著更高，而BSO导致86Rb+摄取水平降低，这表明辐射对Na+/K(+)-ATP酶泵的影响是由ROS引起的。此外，与对照PW细胞相比，暴露于NAC的细胞静息细胞膜电位略有超极化，而与对照PW细胞相比，暴露于BSO的细胞则发生去极化。总之，这些数据表明Bcl-2影响Na+/K(+)-ATP酶泵活性，这与静息膜电位以及对辐射诱导PCD的敏感程度相关。
