Failure of a Second X-ray Dose to Activate Nuclear Factor κB in Normal Rat Astrocytes

Uma Raju,Ruisheng Lu,Florence Noel,Glenice J Gumin,Philip J Tofilon

doi:10.1074/jbc.272.39.24624

Abstract

Induced gene expression and subsequent cytokine production have been implicated in the normal tissue injury response to radiotherapy. However, studies of radiation-induced gene expression have used single radiation doses rather than the fractionated exposures typical of the clinical situation. To study the effects of multiple radiation doses on gene expression, we investigated nuclear factor kappaB (NFkappaB) DNA binding activity in primary astrocyte cultures after one and two exposures to x-rays. After a single dose of x-rays (3.8-15 gray (Gy)), NFkappaB binding activity in astrocytes increased in a dose-dependent manner, reaching a maximum by 2-4 h and returning to control levels by 8 h after irradiation. In split-dose experiments, when an interval of 24 h was used between two doses of 7.5 Gy, the second 7.5-Gy exposure failed to induce NFkappaB activation. The period of desensitization induced by the first radiation exposure was dose-dependent, persisting approximately 72 h after 7.5 Gy compared with 24 h after 1.5 Gy. No changes in IkappaBalpha protein levels were detected. However, the presence of a transcription inhibitor prevented the desensitizing effect of the initial irradiation. Irradiation also prevented NFkappaB activation in astrocytes by a subsequent exposure to H2O2, but it had no effect on the activation induced by tumor necrosis factor-alpha. These data indicate that an initial x-ray exposure can desensitize astrocytes to the NFkappaB-activating effects of a subsequent radiation exposure. Furthermore, they suggest that this desensitization depends on gene transcription and may have some specificity for NFkappaB activation mediated by reactive oxygen species.

Highlights

It is well established that ionizing radiation kills cells and alters the expression of specific genes in surviving cells
Both the upper and lower bands representing nuclear factor ␬ B (NF␬B) binding activity increased after irradiation (Fig. 1A), because of the variability in detecting the less intense lower band in unirradiated cultures, only the upper band was used to quantitate the relative increase in binding activity after radiation exposure
When the tumor cells were first exposed to 7.5 Gy followed by a second dose of 15 Gy 24 h later, the level of NF␬B activation induced was the same as that observed with a single dose of 15 Gy. These data indicate that, in contrast to astrocytes, irradiation of the U-373 MG cells does not result in a refractory period. Because of their involvement in a variety of central nervous system (CNS) disease states and their increased expression after other types of CNS damage, cytokines have been suggested to play a role in the pathogenesis of radiation-induced CNS injury [1]

Summary

Introduction

It is well established that ionizing radiation kills cells and alters the expression of specific genes in surviving cells. Among the functional consequences of radiation-induced modulation in gene expression is the increased production of certain cytokines, which has been suggested to be of particular relevance to the normal tissue injury that can result from radiotherapy for cancer [1,2,3,4]. Radiation-induced gene expression and subsequent cytokine production may play a critical role in determining the response of normal tissue to radiotherapy. At present, it is unclear whether the induction of a given cytokine in irradiated cells contributes to a tissue-specific recovery process or is involved in the pathogenesis of radiation-induced normal tissue injury. Type 1 astrocytes are the most prevalent cell type within the central nervous system (CNS) and are a phosphate-buffered saline; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; Gy, gray

Methods

Results

Conclusion