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

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 κB (NFκB) 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)), NFκB 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 NFκB 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 IκBα protein levels were detected. However, the presence of a transcription inhibitor prevented the desensitizing effect of the initial irradiation. Irradiation also prevented NFκB activation in astrocytes by a subsequent exposure to H2O2, but it had no effect on the activation induced by tumor necrosis factor-α. These data indicate that an initial x-ray exposure can desensitize astrocytes to the NFκB-activating effects of a subsequent radiation exposure. Furthermore, they suggest that this desensitization depends on gene transcription and may have some specificity for NFκB activation mediated by reactive oxygen species.

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 B (NFB) 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)), NFB 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 NFB 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 IB␣ protein levels were detected. However, the presence of a transcription inhibitor prevented the desensitizing effect of the initial irradiation. Irradiation also prevented NFB activation in astrocytes by a subsequent exposure to H 2 O 2 , but it had no effect on the activation induced by tumor necrosis factor-␣. These data indicate that an initial x-ray exposure can desensitize astrocytes to the NFB-activating effects of a subsequent radiation exposure. Furthermore, they suggest that this desensitization depends on gene transcription and may have some specificity for NFB activation mediated by reactive oxygen species.
It is now well established that ionizing radiation not only kills cells but also 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). After irradiation of normal cells and tissues, genes reported to be induced include those coding for TNF-␣ 1 (5,6), interleukin-1␤ (6), interleukin-6 (7), transforming growth factor-␤ (2,4), and basic fibroblast growth factor (8). These cytokines have a wide variety of cell type-dependent biological effects, and increases in their production can be expected to influence cells both inside and outside the radiation field. Thus, 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 actually involved in the pathogenesis of radiation-induced normal tissue injury. Regardless, modulation of cytokine activity, whether through replacement therapy or the use of inhibitors, represents a potential strategy for the amelioration of the normal tissue injury that can result from radiotherapy.
Investigations of radiation-induced gene expression in normal cells and tissues to date have focused primarily on the effects of single doses. In a clinical setting, however, radiotherapy is delivered in multiple fractions. With respect to clonogenic cell death and multiple doses, each dose of radiation can be expected to result in at least the same proportional reduction in cell survival (9). Regarding induced gene expression, whether the effect of a subsequent radiation dose is the same as that resulting from the first dose has not, to our knowledge, been determined. A reasonable subject for such a study is the transcription factor nuclear factor B (NFB), which plays a major role in regulating genes coding for proteins involved in inflammation and immunity (10). In normal cells, ionizing radiation has been shown to activate NFB in lymphoid cells (11,12), fibroblasts (7), and, more recently, endothelial cells (13). Furthermore, NFB activation has been found to contribute to TNF-␣ and interleukin 6 gene transcription induced by ionizing radiation (7,14).
Considerable information is available concerning the biochemical regulation of NFB activity (10). In unstimulated cells, NFB is sequestered in the cytoplasm by complexing with the inhibitory protein IB␣. Upon stimulation, IB␣ is degraded and NFB translocates into the nucleus, where it binds to B sites and regulates the transcription of specific genes. One of the genes that has a B site in its promoter is IB␣. Thus the activation of NFB increases the production of IB␣, which then binds to and deactivates free NFB. Because NFB activity is subject to this type of negative feedback control, it might be expected that the activation of NFB by an initial dose of radiation would prevent activation by a second dose.
To test this hypothesis, we evaluated NFB DNA binding in primary cultures of normal rat type 1 astrocytes after one and two doses of x-rays. Type 1 astrocytes are the most prevalent cell type within the central nervous system (CNS) and are a major source of cytokines, proteases, and other types of bioactive molecules (15). This in vitro model has been used extensively to investigate cytokine production by astrocytes under a variety of environmental and treatment conditions. Moreover, many of the signaling pathways regulating the synthesis of specific cytokines, neurotransmitters, and small molecular weight molecules such as nitric oxide defined in astrocyte cultures have been shown to be operative in vivo (16,17). Of particular relevance to radiation studies, astrocytes are evaluated as confluent cultures, thus eliminating the complicating variables of cell cycle delay and redistribution. The data presented here indicate that, although the first dose of radiation increases NFB activity in astrocyte cultures, the initial exposure desensitizes astrocytes to the NFB-inducing actions of a second dose.

MATERIALS AND METHODS
Cell Culture-Primary cultures of type I astrocytes were generated from the cortex of 21-day-old Sprague-Dawley rat embryos as described by McCarthy and de Vellis (18) with some modifications (19). The cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 10 g/ml gentamycin, and 2 mM glutamine in a humidified atmosphere of 5% CO 2 , 95% air at 37°C. After reaching confluence, contaminating oligodendrocytes and microglia were removed by overnight shaking followed by exposure to 20 M cytosine arabinoside for two periods of 48 h with an interval of 1 day between treatments. After the second cytosine arabinoside exposure, the cultures were maintained in DMEM containing 10% FBS for 1 day and then fed with DMEM containing 2% FBS. The cultures were maintained for at least 2 days in DMEM with 2% FBS before use in an experiment. The human glioma cell line U-373 MG (obtained from ATCC, Rockville, MD) was grown in DMEM containing 10% FBS until confluent and then maintained in 0.5% FBS for 1 day before use in an experiment.
Culture Treatment-Irradiation was performed using a Philips RT-250 x-ray unit with a 1-mm copper filter at the dose rate of 176 cGy/min. Cultures were returned to the incubator after irradiation, and cells were harvested at specified times. In experiments involving two x-ray exposures, astrocyte cultures were irradiated and returned to the incubator for a specified period of time; a second dose of radiation was then delivered, and cultures were again returned to the incubator for 2 h before analysis. In some experiments, cultures were treated with TNF-␣ (1 nM, 1 h), H 2 O 2 (100 M, 1 h), or 5,6-dichlorobenzimidazole riboside (DRB) (100 M, 5 h).
Preparation of Nuclear Extracts-Cells were scraped from tissue culture flasks in 10 ml of ice-cold phosphate-buffered saline and pelleted by centrifugation at 1500 ϫ g for 5 min. The cell pellet was resuspended in 400 l of ice-cold lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, and 1 g/ml pepstatin A). After incubation on ice for 15 min, 12 l of 10% Nonidet P-40 were added. Samples were then vigorously vortexed for 10 s and centrifuged for 30 s in a microcentrifuge. The nuclear pellet was resuspended in 30 l of ice-cold extraction buffer (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, 2 g/ml leupeptin, and 1 g/ml pepstatin A), incubated with slow rocking at 4°C for 30 min, and then centrifuged for 5 min. The supernatant (nuclear extract) was collected and frozen at Ϫ80°C. Protein concentration was determined using a Bio-Rad DC kit (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)-EMSA was performed using the gel shift assay system from Promega (Madison, WI) as described in the protocol provided by the manufacturer with some modifications. Briefly, the oligonucleotide containing an NFB consensus sequence (5Ј-␣GT TGA GGG GAC TTT CCC AGG C-3Ј) was end-labeled with T4 polynucleotide kinase and [␥-32 P]ATP. The probe (2 l) and the nuclear extracts (5 g) were incubated with or without competitor in the presence of 10 mM Tris-HCl (pH 7.5), 1 mM MgCl 2 , 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, and 50 ng/ml poly(dI-dC) in 16 l total volume at 37°C for 15 min. The reaction was stopped with 1 l of gel loading buffer and resolved in a 5% native polyacrylamide gel. The gel was then dried, and the radioactivity of the bands was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The supershift analysis was performed essentially the same as EMSA, except that antibodies were incubated with nuclear extracts before the probe was added. The antibodies to NFB p50 (NLS), NFB p65 (C-20), and c-Fos (K-25) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Western Blotting-Astrocyte cultures were washed once with ice-cold PBS, scraped into 10 ml of PBS, and pelleted. The cell pellet was resuspended in 3 volumes of lysis buffer (50 mM Tris-HCl, pH 8.0, 400 mM NaCl, 5 mM EDTA, 5 g/ml aprotinin, 5 g/ml leupeptin, 20 mM NaF, and 1% Nonidet P-40) were added; the suspension was then incubated on ice for 20 min and vortexed for 10 s. The cell lysate was spun at 12,000 ϫ g for 10 min at 4°C. Aliquots of the resulting supernatant were stored at Ϫ80°C. Protein concentration was determined using the Bio-Rad DC protein assay kit. Whole cell lysates (30 g) were applied to a 6% stacking, 10% SDS-polyacrylamide gel and resolved at 60 mA constant current for 40 min. After electrophoresis, the gel was electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham Corp.). The nonspecific sites on the membrane were blocked at 4°C overnight with 5% nonfat milk in PBS supplemented with 0.1% Tween 20 (PBS-T). The membrane was washed with PBS-T and probed with rabbit anti-rat IB␣ polyclonal antibody (C-21, Santa Cruz Biotechnology) at 1.0 g/ml in the blocking mix for 45 min. The membrane was washed in PBS-T and incubated with donkey anti-rabbit IgG-horseradish peroxidase (Amersham) at a 1:2000 dilution in the blocking mix. The membrane was washed with PBS-T, and detection was performed using ECL Western blotting detection reagents (Amersham). The membrane was then exposed to Hyperfilm-ECL (Amersham), and the image scanned with a Molecular Dynamics scanning densitometer.

RESULTS
To characterize the effects of a single x-ray dose on NFB activity in astrocytes, an EMSA was performed using an oligonucleotide containing a B site and nuclear extracts from control and irradiated primary astrocyte cultures (Fig. 1). Using this oligonucleotide, three bands were detected (Fig. 1A). To determine which bands corresponded to NFB binding, 50-fold unlabeled oligonucleotide was included into the reaction mixture (Fig. 1A, lane 2). The two upper bands disappeared in the presence of the unlabeled oligonucleotide indicative of specific NFB binding. The lowest band, however, was reduced only slightly under these conditions and was considered to reflect primarily nonspecific binding; the intensity of the nonspecific, lowest band was not altered by irradiation. After a 15-Gy exposure, NFB binding activity in astrocytes increased in a time-dependent manner. An increase in binding was apparent in the upper two bands at 1 h, reaching a maximum of about a 4-fold induction at 2 h after irradiation and returning to the control level by 8 h. The dose dependence of x-ray-induced NFB DNA binding activity was determined in astrocyte cultures 2 h after irradiation (Fig. 1B). Although both the upper and lower bands representing NFB 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. Irradiation with 1.5 Gy had no detectable effect on NFB binding activity, but beginning at 3.8 Gy, a dose-dependent increase was induced that extended up to at least 15 Gy. These results illustrate that x-rays induce NFB DNA binding activity in astrocytes in a dose-and time-dependent manner. It should be emphasized that the astrocyte cultures were confluent at the time of irradiation and thus were not subject to the complicating variables of cell cycle redistribution or the expression of clonogenic cell death. Furthermore, no significant cytotoxicity can be detected out to at least 48 h after 20 Gy (19). Thus, the NFB activation observed after astrocyte irradiation occurs in a biologically relevant dose range.
Supershift analysis using antibodies to p65 and p50 indicated that the lower band representing NFB binding activity was composed of p50 homodimers, whereas the upper band consisted of a mixture of p65 homodimers and p65/p50 heterodimers (Fig. 2). The presence of p65 homodimers is consistent with a previous report identifying their induction in astro-cyte cultures treated with TNF-␣ (20). The c-Fos antibody was included in this analysis as a control for nonspecific interactions.
The effect of a second radiation exposure on astrocyte NFB activity was investigated using a split-dose protocol similar to those traditionally used in the analysis of clonogenic cell survival (9). In the study shown in Fig. 3, 15 Gy were divided into two 7.5-Gy doses delivered at an interval of 24 h, a time at which the induced NFB DNA binding resulting from a single radiation exposure has returned to control levels (see Figs. 1A and 3, lane 6). Nuclear protein extracts were prepared 2 h after the second irradiation and NFB DNA binding activity was determined. Irradiation of astrocytes with single doses of 7.5 and 15 Gy analyzed at 2 h resulted in approximately 2.5-and 4-fold increases in NFB binding activity, respectively. However, when 7.5 Gy were followed 24 h later with an additional 7.5 Gy, no significant NFB activation was detected after the second dose. This loss of activation cannot be attributed to merely a delay in the time course, because the same lack of induced NFB DNA binding was observed when analyzed out to 8 h after the second irradiation (data not shown). These data suggest that after irradiation there is a refractory period during which the susceptibility of NFB to activation by a subse- To investigate the dose requirements for inducing this refractory period, astrocytes were exposed to 1.5, 3.8, or 7.5 Gy followed at specified intervals by a second dose of 15 Gy. Nuclear protein extracts were prepared 2 h after the second irradiation, and NFB DNA binding activity was determined. A representative EMSA obtained after irradiation of astrocytes with 1.5 Gy followed by a subsequent dose of 15 Gy after specified intervals is shown in Fig. 4. As in previous experiments, a single dose of 15 Gy increased NFB binding approximately 4-fold. However, when the 15 Gy were preceded 1-8 h beforehand with a 1.5-Gy exposure, the increase in NFB activity was markedly reduced. This reduction was maximum when 15 Gy were delivered 2 h after the initial 1.5-Gy exposure, at which time no radiation-induced NFB activity was detected. In contrast to the data shown in Fig. 3, indicating that after 7.5 Gy the refractory period lasted for at least 24 h, by 4 h after the initial dose of 1.5 Gy, the susceptibility of NFB to activation by 15 Gy began to return to single exposure levels. The influence of radiation dose on the length of the refractory period is summarized in Fig. 5. After initial doses of 7.5, 3.8, or 1.5 Gy, the susceptibility of NFB to activation by 15 Gy returned by approximately 72, 36, and 24 h, respectively. These data indicate that the ability of radiation to induce a refractory period or to desensitize astrocytes to NFB activation by a subsequent irradiation is dose-dependent.
To determine whether gene transcription is necessary for this radiation-induced desensitization, the two radiation exposures protocol shown in Fig. 4 (1.5 Gy followed by 15 Gy) was performed in the presence of the transcription inhibitor DRB. In contrast to actinomycin D, DRB does not intercalate into nucleic acids, but inhibits RNA polymerase II (21)(22)(23)(24). This results in a reversible and relatively rapid inhibition of RNA synthesis. Treatment of astrocytes with DRB (100 M) for 1 h inhibits the incorporation of [ 3 H]uridine into RNA by 90% (data not shown). Astrocytes were exposed to DRB for 1 h before irradiation with 1.5 Gy; 2 h later, the cultures received 15 Gy, and 2 h after that, the nuclear extract was examined for NFB activity. DRB was present during the entire treatment protocol. Three independent experiments were performed, and a representative EMSA is shown in Fig. 6. DRB treatment of astrocytes alone (a total of 5 h) induced a slight increase in NFB activity (2.3 Ϯ 1.5-fold, mean Ϯ S.E., n ϭ 3), which is probably due to the inhibition of IB␣ synthesis, as has been suggested in studies using cycloheximide (25). Pretreatment of astrocytes with DRB had no effect on NFB activation induced by a single exposure to 15 Gy (4.4 Ϯ 0.6 versus 4.5 Ϯ 0.9 for 15 Gy without DRB). In the absence of DRB, 15 Gy failed to activate NFB when administered after the initial radiation exposure to 1.5 Gy (lane 4), which is consistent with the data shown in Fig. 4. However, in the presence of DRB, the desensitization was eliminated, and the second radiation exposure induced NFB activation (lane 5). The activation resulting from the combination of DRB and 1.5 Gy followed by 15 Gy is 9.4 Ϯ 2.8-fold over untreated levels, which is greater than the activation induced by a single dose of 15 Gy (4.5 Ϯ 0.9-fold). It is not possible at this time to determine whether the effect of the combined radiation protocol and DRB is additive or supraadditive. However, these data do indicate that induced gene expression resulting from the first x-ray exposure is required for the desensitization of NFB activation to the subsequent irradiation.
The requirement for gene induction suggested that increases in IB␣ levels may be involved in the refractory period after irradiation. It has recently been shown that, in HeLa and lymphoid cell cultures, glucocorticoids inhibit NFB activity through the induction of IB␣ synthesis (26,27). To determine whether such a scenario might account for the failure of a second radiation dose to increase NFB activity, IB␣ protein levels were determined by Western blot analysis 24 h after irradiation of astrocyte cultures with 1.5 or 7.5 Gy. At this time after the first 7.5 Gy, NFB activation by a second radiation exposure is essentially eliminated (see Fig. 4). If IB␣ was responsible for the ineffectiveness of the second radiation exposure, then the level of this protein would be expected to be elevated at this time. As shown in Fig. 7, the IB␣ protein level in cultures irradiated with 1.5 or 7.5 Gy was the same as in the unirradiated cultures. Northern blot analysis showed that the IB␣ mRNA level was also not affected under these conditions (data not shown). These results suggest that the synthesis of IB␣ is unlikely to be involved in the desensitizing actions of the initial irradiation.
NFB DNA binding is susceptible to induction by a wide variety of stimuli. To determine whether the desensitizing effect also occurs after other activators of NFB, astrocyte cultures were treated with various combinations of x-rays, H 2 O 2 , and TNF-␣. H 2 O 2 (100 M, 1 h) induced NFB DNA binding with the maximum activation at 4 h post-treatment and returning to untreated levels by 24 h (data not shown). As shown in Fig. 8 (Table I). Thus, H 2 O 2 and ionizing radiation appear to have similar desensitizing effects. A different situation, however, existed for TNF-␣. Treatment of astrocytes with TNF-␣ (1 nM, 1 h) induced NFB activation by 7-fold, which returned to untreated levels by 24 h post-treatment (data not shown), consistent with previous results (19). In contrast to H 2 O 2 , an initial exposure of astrocytes to 7.5 Gy 24 h before TNF-␣ treatment had no effect on the NFB activating capability of TNF-␣ (Fig. 8). When cells were first treated with TNF-␣ and then exposed to 15 Gy 24 h later, a partial desensitization to radiation activated NFB resulted (Table I). Interestingly, when TNF-␣ was followed 24 h later by a second TNF-␣ exposure, the NFB activation was essentially the same as that induced by the first treatment. These data suggest that there is some specificity regarding the stimuli that are capable of inducing NFB desensitization in astrocytes. On a technical note, the failure of radiation to alter NFB activation by a subsequent TNF-␣ exposure indicates that the initial irradiation does not modify the solubility of the NFB complex such that the efficiency of the extraction procedure used in

TABLE I Effects of combinations of x-rays, H 2 O 2 , and TNF-␣ on NFB
activation in astrocyte cultures Cells were exposed to an initial dose of 7.5 Gy, H 2 O 2 (100 M, 1 h) or TNF-␣ (1 nM, 1 h) followed 24 h later by the second specified treatment. Nuclear protein extraction and EMSA were performed as described under "Materials and Methods," and binding activity was quantitated using a PhosphorImager. Results are presented as the fold stimulation compared with untreated cultures. Values represent the mean Ϯ S.E. of three to four independent experiments except for TNF-␣ followed by TNF-␣, which was performed only twice. these studies is significantly affected. Ionizing radiation has been reported to activate NFB DNA binding in a number of cell types. To determine whether the radiation-induced refractory or desensitization period is unique to astrocyte cultures, the human glioma cell line U-373 MG was exposed to two doses of x-rays (7.5 and 15 Gy) and NFB DNA binding activity was analyzed (Fig. 9). A single exposure to 15 Gy activated NFB approximately 4-fold, with induction being highest 4 h after radiation and returning to control levels by 24 h. When the tumor cells were first exposed to 7.5 Gy followed by a second dose of 15 Gy 24 h later (the same protocol as for astrocyte cultures), the level of NFB 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. DISCUSSION Because of their involvement in a variety of 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). Indeed, Hong et al. (6) recently showed that the expression of TNF-␣, interleukin1␤, and I-CAM-1 are increased in the mouse brain after a single dose of radiation. It was hypothesized that these inflammation/immunity-related gene products may also play a role in radiation-induced CNS damage either directly or through the initiation of a cellular and/or molecular cascade of events. A transcription factor critical to the regulation of genes coding for products participating in defense-type reactions such as inflammation and immunity, including those mentioned above, is NFB (10). As shown herein, irradiation of astrocytes in vitro significantly increased NFB DNA binding activity, which lends support to the concept that induced gene expression may be involved in the radioresponse of the CNS, at least after a single dose. The results of our study, however, also indicate that irradiation of normal astrocytes results in a refractory period during which the susceptibility of NFB to activation by a subsequent x-ray exposure is significantly reduced.
Although the response of most normal tissues to multiple radiation treatments has been described in experimental models, investigations of the pathogenesis of radiation-induced normal tissue injury have focused primarily on the use of single doses. This also applies to studies specifically addressing radiation-induced gene expression. Experimental convenience and the necessity for a starting point for these relatively new investigations are certainly valid rationales for focusing on single radiation exposures. However, there is no a priori reason to assume that the changes in gene expression induced in cells and tissues after the first dose of radiation will be the same as those induced by a subsequent exposure. The data presented herein indicate that in the delivery of two radiation doses to normal astrocytes in vitro, using a clinically relevant interval of 24 h, the first dose markedly affects the response of the cells to the second x-ray dose. If a similar desensitization process occurs in vivo, then the spectrum of genes induced by the first radiation dose would not be expected to be induced by a subsequent dose. Thus, to understand the role of radiation-induced gene expression in the pathogenesis of normal tissue injury produced by radiotherapy, it will be necessary to define this event as it occurs after more than one radiation exposure.
Although the mechanism responsible for the desensitization induced by radiation has not been completely defined, our initial studies do provide some insight. As shown in Figs. 5 and 6, the length of the refractory period after irradiation of astrocytes is dose-dependent, and its induction requires gene transcription. These two observations raise the possibility that the desensitization involves the increased production of a protein that inhibits radiation-induced NFB activation. Auphon et al. (26) and Scheinman et al. (27) have shown that the glucocorticoid-mediated inhibition of NFB activity involves the induction of IB␣ synthesis. In astrocytes, elevated IB␣ levels, however, were not detected after irradiation, suggesting that an increased sequestration of free NFB by IB␣ is unlikely to be responsible for the ineffectiveness of a second radiation exposure. Although gene transcription may be required, the refractory period in astrocytes was induced by exposure to as little as 1.5 Gy, which does not induce a detectable level of NFB DNA binding (Fig. 2). This low dose effect suggests that the desensitization may not require NFB activation and, consequently, may not involve the induction of B regulated genes. This possibility is consistent with the lack of a refractory period detected after TNF-␣ treatment (see below).
Additional information pertaining to the mechanism of radiation-induced desensitization is provided from the studies combining x-rays with other activators of NFB. A critical regulatory step in NFB activation is the dissociation of the NFB/ IB complex (28). Brown et al. (29) showed that the mutation of IB␣ at the Ser 32 residue, a potential phosphorylation site, can prevent NFB activation. Thus, the mechanism responsible for the radiation-mediated desensitization of astrocytes could involve some change in the NFB/IB complex that prevents its dissociation. Prior irradiation of astrocyte cultures, however, did not affect NFB activation by TNF-␣, indicating that the dissociation process of NFB from IB␣ is fundamentally intact in irradiated astrocytes. Furthermore, the TNF-␣-mediated activation of NFB in irradiated astrocytes and in cultures previously exposed to TNF-␣ suggests that an upstream event specific to a radiation-activated signal transduction pathway is responsible for the failure of a second x-ray dose to activate NFB. H 2 O 2 and x-rays are essentially interchangeable regard- FIG. 9. Effect of two x-ray exposures on NFB DNA binding activity in the glioma cell line U-373 MG. Cultures were exposed to an initial dose of 7.5 Gy followed by a second dose of 15 Gy 24 h later. NFB DNA binding activity was determined 4 h after the 15 Gy exposure. Lane 1, 15-Gy exposed cells that had been incubated with a 50-fold excess of unlabeled probe oligonucleotide (competitor); lane 2, untreated culture; lane 3, 15-Gy exposed cells harvested 4 h later; lane 4, 7.5-Gy exposed cells harvested 24 h later; lane 5, 7.5-Gy followed by 15-Gy exposed cells harvested 4 h later. Brackets indicate specific DNA-protein complexes; the asterisk (*) indicates a nonspecific complex.
ing the induction of the desensitization (Table I); both act to increase the intracellular levels of reactive oxygen species. The differences in the desensitizing actions of x-rays/H 2 O 2 and TNF-␣ thus suggest that the pathway mediating NFB activation by reactive oxygen species is susceptible to desensitization as compared with the activation that proceeds through a receptor-mediated stimulus such as TNF-␣. These results also support the notion of separate pathways for oxidant-initiated and redox-regulated activation of NFB (30).
It is well established that radiation can activate NFB binding in a number of cell types; however, whether the desensitization effect detected in astrocytes is induced in other cells remains to be determined. As shown in Fig. 9, radiation activated NFB binding in a human brain tumor cell line with similar kinetics as in astrocytes, and yet, exposure to a second dose of x-rays induced the same level of NFB activation as the first dose. Thus, a refractory period was not induced in these brain tumor cells. Obviously, there are many caveats in attempting to compare the response of an established human brain tumor cell line to that of a primary culture of normal rat cells; other cell types need to be evaluated, especially those of normal origin. However, these results indicate that the desensitization of radiation-induced NFB activation does have at least some dependence on cell type. Futhermore, if the lack of a desensitization process can be attributed to brain tumor cells in general, it may suggest that these tumor cells, in contrast to normal astrocytes, continue to produce NFB-dependent cytokines during fractionated radiotherapy. Such cytokines may thus continue to influence the growth rate and invasion propensity of the tumor as well as the immune response of the host.