It has been suggested that the cellular response to exposure to ionizing radiation involves activation of the transcription factor nuclear factor-κB (NF-κB) and that this response is defective in cells from individuals with ataxia telangiectasia (AT). In one study, it was found that SV40 large T-transformed cells derived from a patient null for the AT mutated (ATM) gene exhibited constitutive activation of NF-κB and that in those cells, inhibition of NF-κB by expression of a modified form of IκBα led to correction of the radiosensitivity associated with the AT phenotype [M. Jung et al., Science (Washington DC), 268: 1691–1621, 1995]. From those data, it was suggested that NF-κB played a role in the AT phenotype. We show here that normal diploid cells derived from AT patients do not exhibit constitutive activation of NF-κB. Furthermore, we provide data that the transformation process associated with SV40 large T antigen expression in AT−/− cells leads to aberrant cellular responses. Our studies highlight the importance of using diploid, nontransformed AT−/− cells for in vitro studies relevant to the AT phenotype whenever possible.

AT3 is a rare human autosomal recessive disorder characterized by a wide variety of pleiotropic defects in multiple systems. AT patients suffer from growth retardation, neuronal degeneration in the cerebellum leading to ataxia, dilated blood vessels in the eye and facial area, gonadal defects, immunodeficiencies, a high incidence of cancer, and hypersensitivity to IR (1). Cells derived from AT patients also exhibit a variety of abnormalities including cytoskeletal defects, hypersensitivity to IR, higher requirements for serum growth factors, and a reduced life span in culture (1). In addition, AT cells exhibit radioresistant DNA synthesis (1) and display defects in G1-S and G2-M checkpoints in response to IR (2).

The gene responsible for AT has been cloned and was designated ATM(3, 4). The open reading frame of the ATM transcript encodes a protein of 3056 amino acids with a predicted molecular mass of 350 kDa (3, 4). The ATM protein belongs to an expanding family of large proteins that have a highly conserved COOH-terminal region that shows sequence similarity to the catalytic domain of phosphatidylinositol 3-kinases (1). These proteins have all been shown to be involved in the regulation of cell cycle progression and checkpoint responses to DNA damage (1). Therefore, it is commonly thought that ATM plays a vital role in transducing the DNA damage signal to the repair machinery. Further evidence for this comes from recent studies that show that ATM phosphorylates the p53 tumor suppressor protein in response to IR, and that this phosphorylation is absent or delayed in AT fibroblasts (5, 6).

The transcription factor NF-κB plays an important role in initiating immune and inflammatory responses (reviewed in Ref. 7). In addition, NF-κB activation protects cells from apoptosis induced by a number of different stimuli, including IR and radiomimetic drugs (8). There are five known members of the mammalian NF-κB/Rel family: p65 (RelA), c-Rel, RelB, p50 (NF-κB1), and p52 (NF-κB2). NF-κB exists as a homo- or heterodimer with various combinations of these subunits. Classic NF-κB is a p50-p65 heterodimer and typically resides in the cytoplasm in an inactive form, bound by its inhibitory proteins, members of the IκB family (reviewed in Ref. 9). Upon stimulation with various agents, signal transduction events result in phosphorylation, ubiquitination, and degradation of IκB (10) and the subsequent release of NF-κB, which translocates into the nucleus and regulates gene expression.

IR has been shown to be an inducer of NF-κB in certain cell types (11, 12, 13, 14). Because NF-κB has been shown to protect cells from apoptosis in response to IR (8), NF-κB may, therefore, be a critical factor in regulating the cellular response to IR. Previous work has shown that one line of SV40 large T-immortalized fibroblasts from an AT patient has constitutively high levels of nuclear NF-κB and expresses high amounts of the IκBα transcript (15), a gene that is regulated by NF-κB. Furthermore, expression of a truncated form of IκBα, missing the first 45 NH2-terminal amino acids, was able to correct the radiation sensitivity of these SV40 large T-transformed AT cells (15). Expression of this truncated form of IκBα was also able to restore regulated activation of NF-κB in response to IR. Thus, it was concluded that the loss of ATM function led to the activation of NF-κB, which somehow promoted sensitivity to radiation. Evidence from our lab and other labs has shown that cellular transformation, including expression of certain viral oncoproteins, can alter the activity of NF-κB (16, 17, 18, 19). In addition, a recent report demonstrated that SV40 large T-transformed AT fibroblasts undergo apoptosis in response to IR, whereas nontransformed AT fibroblasts do not (20). Therefore, because cellular transformation alters both NF-κB activity and the response of AT cells to IR, we decided to determine whether the loss of ATM in fibroblasts isolated from patients with AT affected the regulation of NF-κB. Our findings indicate that NF-κB regulation is not affected in nontransformed fibroblasts isolated from patients with AT. Furthermore, IR is not able to induce activation of NF-κB in either normal diploid human fibroblasts or in the diploid fibroblasts from AT patients. In contrast to the SV40 large T-transformed AT fibroblasts, the nontransformed diploid AT fibroblasts do not exhibit a constitutively high level of nuclear NF-κB, nor do they express high levels of the IκBα transcript.

Cells and Treatments.

Nontransformed dermal fibroblasts from AT-affected individuals (GM02052, GM03395, AG03058, and GM05823), AT dermal fibroblasts from an obligate AT heterozygote (GM03396), fibroblasts from normal individuals (GM03349), and an SV40-immortalized cell line (GM05849) derived from the strain GM05823 were obtained from the Human Genetic Mutant Cell Repository and the Aging Cell Repository (Coriell Cell Repositories, Camden, NJ). NHFs were obtained from M. Cordiero-Stone (University of North Carolina-Chapel Hill). Cells were grown in Eagle’s MEM with 20% fetal bovine serum, 2× l-glutamine, and 2× serine, aspartic acid, and pyruvic acid. Cells were used between passage numbers 10 and 20. Exposure to IR was performed using a 137Cs source of γ-rays at a dose rate of ∼1 Gy/min, and cells were harvested at the indicated time points after exposures. Treatment of cells with TNF-α (Promega Corp., Madison, WI) was performed for 30 min at a final concentration of 10 ng/ml.

Nuclear and Cytoplasmic Extracts.

After treatments, nuclear and cytoplasmic extracts were made by washing the cells with ice-cold PBS and gently scraping them from the plates. The cells were then transferred to microcentrifuge tubes and lysed on ice in two pellet volumes of cytoplasmic extraction buffer [10 mm HEPES (pH 7.6), 60 mm KCl, 1 mm EDTA, 0.3% NP40, 1 mm DTT, 1 mm PMSF, and 2.5 μg each of aprotinin, leupeptin, and pepstatin per ml]. Nuclei were pelleted (200 × g at 4°C for 5 min), and cytoplasmic supernatants were transferred to fresh tubes and maintained on ice. Nuclei were washed gently with 100 μl of cytoplasmic extraction buffer without NP40 and pelleted (200 × g at 4°C for 5 min), and the supernatants were discarded. Two pellet volumes of nuclear extraction buffer [20 mm Tris (pH 8.0), 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm PMSF, 25% glycerol, and 2.5 μg each of aprotinin, leupeptin, and pepstatin per ml] were added, and the final salt concentration was adjusted to ∼400 mm with 5 m NaCl. Nuclear pellets were resuspended by vortexing, and the nuclear lysates were maintained on ice for 10 min with occasional vortexing. All cytoplasmic and nuclear extracts were cleared (16,000 × g for 4°C at 10 min) and transferred to fresh tubes. Glycerol was added to the cytoplasmic extracts to a final concentration of 20%. Protein concentrations were determined by the Bradford assay with the Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, Calif.), and all extracts were stored at −70°C.

EMSAs.

For EMSAs, equal amounts of nuclear extracts (5 μg of protein) were incubated for 15 min at room temperature with a 32P-labeled probe containing a NF-κB site from the class I MHC promoter (5′-CAGGGCTGGGGATTCCCCATCTCCACAGTTTCACTTC-3′; the NF-κB binding site is in boldface) in binding buffer [10 mm Tris (pH 7.7), 50 mm NaCl, 0.5 mm EDTA, 1 mm DTT, and 10% glycerol] plus 2 μg of poly(deoxyinosinic-deoxycytidylic acid) (Pharmacia Biotech, Piscataway, NJ). Complexes were fractionated in 5% polyacrylamide gels in high ionic strength Tris-glycine-EDTA buffer (25 mm Tris, 190 mm glycine, and 1 mm EDTA), dried, and autoradiographed.

Western Blot Analysis.

Whole-cell extracts were prepared by lysing cells in buffer containing 50 mm HEPES (pH 7.0), 250 mm NaCl, 0.1% NP40, 5 mm EDTA, 0.5 mm DTT, 1 mm NaF, 1 mm Na3VO4, 1 mm PMSF, and 5 μg/ml each of aprotinin, leupeptin, and pepstatin. Equal amounts of whole-cell extracts were fractionated in a 10% polyacrylamide-SDS gel and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were probed with antibody specific for p65 (Rockland, Gilbertsville, PA) and IκBα (sc-371; Santa Cruz Biotech, Santa Cruz, CA).

Northern Blot Analysis.

mRNA was harvested from cells using RNeasy mini prep kit (Qiagen) according to the manufacturer’s recommendations. Twenty-five μg of mRNA were fractionated on a 1.2% agarose gel containing formaldehyde and was transferred to GeneScreen Plus nylon membrane (NEN Life Science Products, Boston, MA). The membranes were prehybridized in QuickHyb solution (Stratagene, La Jolla, CA). Probes specific for IκBα and glyceraldehyde-3-phosphate dehydrogenase were labeled by nick translation and added directly to the QuickHyb solution. Specific mRNA bands were visualized by autoradiography.

p34CDC2/Cyclin B Histone H1 in Vitro Kinase Assay.

The p34CDC2/cyclin B histone H1 in vitro kinase assays were done as described previously (2). Fifty mg of protein was used per kinase reaction, and 0.5 μl of antihuman cyclin B mixed mouse monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY) was used to immunoprecipitate cyclin B and associated proteins. All kinase assays were done in triplicate and quantified using Molecular Dynamics Phosphorimager and Image-Quant software.

To determine whether ATM plays a role in NF-κB activation, nontransformed normal and AT diploid fibroblast cultures were used to study the response of NF-κB after treatment of cells with IR. To do this, cell cultures were exposed to IR and harvested at the indicated time points after exposure. Nuclei were harvested and used in EMSAs to measure activation of NF-κB. As shown, an untreated normal human fibroblast culture has a modest basal level of nuclear NF-κB (Fig. 1, Lane 1). Nuclear extracts harvested 2 and 4 h after treatment with 6 Gy of IR (Fig. 1, Lanes 2 and 3) do not show any significant increase in the amount of nuclear NF-κB. Importantly, a nontransformed AT fibroblast culture exhibited a lower level of basal nuclear NF-κB (Fig. 1, Lane 5), which is not inducible after exposure of the cells to IR (Fig. 1, Lanes 6 and 7). Nuclear extracts harvested 8, 16, and 24 h after exposure of normal and AT fibroblasts to 6 Gy of IR also showed no increase in NF-κB induction (data not shown), demonstrating that NF-κB induction in response to IR is not a delayed response in these cell types. Both the normal human fibroblasts and AT fibroblasts showed normal activation of NF-κB in response to treatment of the cells with TNF-α (Fig. 1, Lanes 4 and 8), indicating that there is not a defect in the ability of these cells to activate NF-κB.

As a control to show that the NHF and AT fibroblast cultures are responding as expected to IR, p34CDC2/cyclin B1 histone H1 in vitro kinase assays were performed. The NHF fibroblast strain exhibited a 71% (SD, 6) suppression of kinase activity 2 h after 6 Gy of IR, whereas the AT fibroblast strain AG03058 exhibited a 39% (SD, 7) suppression of kinase activity after the same treatment. Similarly, p21 was strongly induced at 2 and 4 h after 6 Gy of IR in the NHF cells but not in the AG03058 AT fibroblast culture (data not shown). The NHF cells exhibited normal suppression of p34CDC2/cyclin B histone H1 kinase activity and p21 induction in response to IR, whereas the AT culture had a much weaker suppression of p34CDC2/cyclin B histone H1 kinase activity and failed to induce p21 in response to the same treatment. These results are in agreement with previous findings for AT cells (2).

To demonstrate that the lack of induction of NF-κB in response to IR is not specific to this particular AT cell culture, we tested the IR response in two other AT cell strains (AG03058 and GM03395) as well as one AT heterozygous culture (GM03396) and a normal dermal human fibroblast strain (GM03349). Results from a representative experiment are summarized in Fig. 1, Lanes 9–24. Consistent with the above data, the normal dermal fibroblasts (Fig. 1, Lanes 9–11), AT fibroblasts (Fig. 1, Lanes 13–15 and 17–20), and the AT heterozygous fibroblasts (Fig. 1, Lanes 21–23) each showed a low basal level of nuclear NF-κB and a lack of induction of NF-κB in response to IR. In addition, all four of these strains showed a normal induction of NF-κB in response to treatment with TNF-α (Fig. 1, Lanes 12, 16, 20, and 24).

The results described above are in contrast to a previous report that concluded that the loss of ATM function led to constitutive activation of NF-κB (15). However, this group analyzed nuclear NF-κB in SV40 large T-transformed AT fibroblasts, and we have studied normal diploid AT fibroblasts. We therefore decided to determine whether this difference was due to transformation of the AT fibroblast culture by SV40 large T rather than because of the absence of ATM. To do this, we compared the levels of nuclear NF-κB in nontransformed AT patient fibroblasts (GM05823) with those of the same culture that had been transformed with SV40 large T (GM05849). As shown in Fig. 2, the nontransformed AT fibroblasts have a low basal level of nuclear NF-κB (Lane 1), which is not inducible by treatment with IR (Lanes 2 and 3). In contrast, transformation of this culture with SV40 large T resulted in a significantly higher level of basal nuclear NF-κB (Lane 5) which, like the nontransformed culture, is not inducible by IR (Lanes 6 and 7). A nontransformed normal fibroblast culture is shown as a control (Lanes 9–12). All three cultures show an increase in nuclear NF-κB in response to treatment with TNF-α (Lanes 4, 8, and 12), demonstrating that NF-κB is present and inducible in response to cytokine treatment.

On the basis of the above results, we wanted to determine whether there were any differences in NF-κB protein levels between normal and AT fibroblasts. To examine this, we performed Western analysis on the p65 subunit of NF-κB. Results show that the level of p65 is similar in both the NHF and AT fibroblast cultures (Fig. 3). Moreover, the levels of p65 are unaffected by exposure of both cell cultures to IR (Fig. 3). Activation of NF-κB is preceded by phosphorylation and subsequent degradation of the NF-κB inhibitory protein, IκBα. To determine whether IR induced degradation of the IκBα protein, Western blot analysis was performed on IκBα. The results show that IR did not induce degradation of IκBα at 2 or 4 h after IR (Fig. 3, Lanes 1–3 and 5–7), whereas a 30-min treatment with TNF-α resulted in complete degradation of IκBα (Fig. 3, Lanes 4 and 8). Finally, because the gene encoding the IκBα protein is regulated by NF-κB, any inducer that activates NF-κB will activate expression of the IκBα gene. Therefore, Northern blot analysis was performed to determine whether IR activated expression of the IκBα gene in response to IR. Treatment of the NHF and diploid AT fibroblast cultures with IR did not result in the induction of IκBα gene expression at either 4 or 6 h after IR, whereas a 30-min stimulation of these cultures with TNF-α resulted in the expected increase in IκBα mRNA (Fig. 4).

The results described in this report show that IR treatment of normal human fibroblasts or fibroblasts derived from AT patients does not result in activation of NF-κB as measured by EMSA. However, a higher level of basal NF-κB was observed in SV40 large T-transformed AT fibroblasts as compared with the nontransformed AT fibroblasts and NHFs. In agreement with the mobility shift data, no increase in IκBα mRNA or decrease in IκBα protein was observed in response to IR treatment. Finally, the level of the p65 subunit of NF-κB is similar in both the NHF and AT fibroblasts, and its level was not affected by treatment with IR.

These results show that induction of NF-κB in response to IR does not appear to be an important cellular response in fibroblast cells, because no increase in NF-κB activation was observed after IR doses ranging from 3 to 20 Gy and at time points extending out to 24 h (see Fig. 1 and data not shown). This is in contrast to the response seen in cells of lymphoid and myeloid origin, which show a high level of activation of NF-κB in response to IR within 30 min of treatment and over a wide range of doses (11, 12, 13).4 A recent report has also shown that NF-κB can be activated by a high dose of IR (20 Gy) in HeLa cells, but the response does not appear until about 2 h after exposure of the cells to IR (14), and the significance of this delayed induction at such a high dose of IR is not clear. The conclusions from our study are in contrast to those of a previous report (15) in which a constitutively high level of nuclear NF-κB was described in SV40 large T-immortalized AT fibroblasts. On the basis of our results, it does not appear that the constitutively high level of nuclear NF-κB seen in the previous report is attributable to the AT phenotype but is attributable to the fact that the cells are immortalized with SV40 large T. In support of this, mortal diploid AT fibroblast cells have a very low level of nuclear NF-κB, similar to levels seen in normal fibroblasts; however, in a SV40 large T-immortalized cell line, derived from these same AT fibroblasts, a much higher level of nuclear NF-κB was seen (see Fig. 2).

Diploid fibroblasts derived from patients with AT have an increased sensitivity to IR compared with normal diploid fibroblasts (1). This hypersensitivity to IR is not, however, attributable to increased apoptosis of these cells, because there is no difference in the frequency of apoptosis in primary fibroblasts isolated from patients with AT than in those isolated from nonafflicted individuals at doses of IR of 5 Gy (20) or 10 Gy.5 In contrast, SV40 large T-immortalized AT fibroblasts do show an increased frequency of apoptosis in response to 5 Gy of IR (20), further indicating that cellular transformation by SV40 large T alters the cells response to IR. A dominant-negative form of IκBα, which prevents activation of NF-κB, has been shown to enhance killing of cells by apoptosis in response to a number of different stimuli including TNF-α and radiomimetic drugs (8), presumably because NF-κB provides a protective effect to the cells by activating expression of antiapoptotic genes (21). However, a recent report showed that another dominant-negative form of IκBα was able to protect SV40 large T-transformed AT fibroblast cells from apoptosis in response to IR (22). The reason for this discrepancy is not clear; however, it appears that transformation of AT fibroblasts by SV40 large T causes these cells to undergo apoptosis in response to IR (20). Because expression of the dominant-negative IκBα protects the SV40 large T-transformed cells from apoptosis, it is possible that this form of IκBα may block the transforming effects of SV40 large T. Therefore, in the presence of the dominant-negative form of IκBα, the SV40 large T-transformed AT fibroblasts may respond to IR more like the nontransformed diploid fibroblasts that do not undergo apoptosis in response to IR.

Activation of NF-κB in response to IR in HeLa cells has been shown to be dependent on activation of the IκB kinase, IKK, which is also activated by TNF-α (14). However, a recent report described separate pathways for NF-κB activation in response to IR and TNF-α in SV40 large T-transformed AT fibroblasts (23). In this report, it was shown that NF-κB was induced in response to 20 Gy of IR in SV40 large T-transformed lung fibroblast cells, but no induction of NF-κB was seen in response to the same dose of IR in SV40 large T-transformed dermal AT fibroblasts. Induction of NF-κB in response to TNF-α was similar in both the SV40 large T-transformed normal and AT fibroblast cell lines, indicating separate pathways for IR and TNF-α-induced induction of NF-κB (23). These results are in contrast to results published previously in which constitutively high levels of activated NF-κB were seen in both the SV40 large T-transformed normal and AT fibroblasts (15). In addition, the NF-κB inhibitory protein IκBα was shown to be degraded in response to IR in a normal SV40 large T-transformed fibroblast line, but no degradation of IκBα was seen in large T-transformed AT fibroblasts. Expression of a stably transfected inducible ATM gene in a large T-transformed AT fibroblast cell line was able to restore degradation of IκBα in response to IR. Finally, in an in vitro phosphorylation assay, immunoprecipitated ATM was shown to be able to phosphorylate a GST-IκBα fusion protein. Although the data presented in this report appear convincing, in light of our data, it is not clear how transformation by SV40 large T may be contributing to activation of NF-κB in response to IR and whether SV40 large T may function differently in the absence of ATM.

Whether NF-κB plays any role in the AT disease process is not yet clear; however, our data suggest that at least in mortal diploid AT fibroblasts, NF-κB is not involved. Furthermore, previous reports using SV40 large T-transformed AT cells to study NF-κB may be clouded by the fact that SV40 large T itself appears to activate NF-κB as well as affect the response of these cells to IR. On the basis of our results, IR does not appear to be an inducer of NF-κB in nontransformed fibroblasts. However, because IR is a potent inducer of NF-κB in cells of lymphoid and myeloid origin, it may be possible that the absence of ATM affects the regulation of NF-κB more so in these types of cells than in fibroblast cells. Future experiments using the ATM knockout mice will be aimed at answering these questions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This research was supported in part by a NIH Grant CA72771 (to A. S. B.). B. P. A. is supported by a postdoctoral fellowship from the Cancer Research Institute.

            
3

The abbreviations used are: AT, ataxia telangiectasia; ATM, AT mutated; IR, ionizing radiation; NF, nuclear factor; NHF, normal human fibroblast; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride.

      
4

B. P. Ashburner and A. S. Baldwin, Jr., unpublished results.

      
5

B. P. Ashburner, C. Y. Wang, and A. S. Baldwin, Jr., unpublished data.

Fig. 1.

Effects of IR on NF-κB activation in normal and AT fibroblasts. Nuclear extracts were harvested after the indicated treatments from control (Lanes 1–4, NHF; Lanes 9–12, GM03349), AT homozygous (Lanes 5–8, GM02052; Lanes 13–16, AG03058; Lanes 17–20, GM03395), and AT heterozygous (Lanes 21–24, GM03396) fibroblasts and used in EMSAs. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The p50:p65 heterodimer and p50:p50 homodimer are indicated. NS, nonspecific band.

Fig. 1.

Effects of IR on NF-κB activation in normal and AT fibroblasts. Nuclear extracts were harvested after the indicated treatments from control (Lanes 1–4, NHF; Lanes 9–12, GM03349), AT homozygous (Lanes 5–8, GM02052; Lanes 13–16, AG03058; Lanes 17–20, GM03395), and AT heterozygous (Lanes 21–24, GM03396) fibroblasts and used in EMSAs. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The p50:p65 heterodimer and p50:p50 homodimer are indicated. NS, nonspecific band.

Close modal
Fig. 2.

Effect of SV40 large T transformation on NF-κB activation. Nuclear extracts were harvested from an AT fibroblasts culture (Lanes 1–4, GM05823) or the same AT culture that had been transformed with SV40 large T antigen (Lanes 5–8, GM05849) and a wild-type control fibroblast culture (Lanes 9–12, GM03349) and used in EMSAs. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The p50:p65 heterodimer and p50:p50 homodimer are indicated. NS, nonspecific band.

Fig. 2.

Effect of SV40 large T transformation on NF-κB activation. Nuclear extracts were harvested from an AT fibroblasts culture (Lanes 1–4, GM05823) or the same AT culture that had been transformed with SV40 large T antigen (Lanes 5–8, GM05849) and a wild-type control fibroblast culture (Lanes 9–12, GM03349) and used in EMSAs. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The p50:p65 heterodimer and p50:p50 homodimer are indicated. NS, nonspecific band.

Close modal
Fig. 3.

Western blot analysis to show the effects of IR on the p65 subunit of NF-κB and on the NF-κB inhibitory protein, IκBα. Whole-cell extracts were isolated from wild-type control (Lanes 1–4, NHF) and AT fibroblast cultures (Lanes 5–8, GM02052) after either mock treatment, 2 or 4 h after exposure to 6 Gy of IR, or treatment with 10 ng/ml TNF-α for 30 min. Twenty μg of WCE were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to immunoblot analysis with antibodies to either p65 (upper panel) or IκBα (lower panel).

Fig. 3.

Western blot analysis to show the effects of IR on the p65 subunit of NF-κB and on the NF-κB inhibitory protein, IκBα. Whole-cell extracts were isolated from wild-type control (Lanes 1–4, NHF) and AT fibroblast cultures (Lanes 5–8, GM02052) after either mock treatment, 2 or 4 h after exposure to 6 Gy of IR, or treatment with 10 ng/ml TNF-α for 30 min. Twenty μg of WCE were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to immunoblot analysis with antibodies to either p65 (upper panel) or IκBα (lower panel).

Close modal
Fig. 4.

Northern blot analysis to show the effects of IR on expression of the NF-κB-regulated IκBα gene. Total cellular RNA was isolated from wild-type control (Lanes 1–4, NHF) and AT fibroblasts cultures (Lanes 4–8, GM02052) and used for Northern blot analysis. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The membrane was probed with a probe specific for IκBα message and then stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

Fig. 4.

Northern blot analysis to show the effects of IR on expression of the NF-κB-regulated IκBα gene. Total cellular RNA was isolated from wild-type control (Lanes 1–4, NHF) and AT fibroblasts cultures (Lanes 4–8, GM02052) and used for Northern blot analysis. Cells were either mock treated, exposed to 6 Gy of IR and harvested 2 and 4 h after exposure, or treated with 10 ng/ml TNF-α and harvested after 30 min. The membrane was probed with a probe specific for IκBα message and then stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

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