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[Cancer Research 62, 1213-1221, February 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Activation of Nuclear Factor {kappa}B in Radioresistance of TP53-inactive Human Keratinocytes1

Xufeng Chen, Binghui Shen, Liqun Xia, Alexander Khaletzkiy2, David Chu, Jeffrey Y. C. Wong and Jian-Jian Li3

Departments of Cell and Tumor Biology [X. C., B. S.], and Radiation Research [L. X., A. K., J. Y. C. W., J. J. L.], and Division of Surgery [D. C.], Beckman Research Institute, City of Hope National Medical Center, Duarte California 91010


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many tumors show a mutant or inactive tumor suppressor p53 (TP53) status, and TP53 in the tumor-carrying human papillomavirus (HPV) may be dysfunctional because of inhibition by the viral protein HPV E6. Molecular mechanisms underlying radiation responses and the radiation-induced resistant phenotype in the TP53-inactive tumor have not been well investigated. In the present study, using a human keratinocyte line (HK18) with TP53 inhibited by HPV18 infection, we demonstrated that nuclear factor (NF)-{kappa}B is responsible for a major portion of the radioresistance observed in a cell population (HK18-IR) derived from HK18 cells by fractionated ionizing radiation (FIR; 2 Gy/fraction; total dose, 60 Gy). HK18-IR cells showed increased clonogenic radioresistance [dose-modifying factor (DMF), 1.47], reduced apoptotic response, and a shortened radiation-induced growth delay. Both DNA-binding and reporter transcriptional activity of NF-{kappa}B, but not of TP53, were activated in HK18-IR cells compared with the parental HK18 cells; this activation was observed both before and after a single dose of 5 Gy. To determine target genes responsive to NF-{kappa}B activation, DNA microarray profiles for 588 genes were matched in HK18-IR cells compared with those in HK18 cells; the paired comparisons were made for basal levels before irradiation or for levels 24 h after 5 Gy. For 25 genes, a 2- to 5-fold up-regulation in HK18-IR cells relative to HK18 cells was similar when comparisons were made for basal levels or for levels after irradiation. Included in the ~4% of genes activated in HK18-IR cells, were six genes (Cyclin B1, Cyclin D1, HIAP, BAG-1, TTF, and fibronectin) putatively linked to NF-{kappa}B regulation. We then measured the expression of this group of FIR-regulated genes in HK18-IR cells expressing a dominant-negative mutant I{kappa}B (mI{kappa}B) that inhibited NF-{kappa}B activation. Clonogenic radioresistance was reduced greatly in the mI{kappa}B transfectants (DMF, 1.18 and 1.10, respectively, at 10% and 1% of isosurvival for mI{kappa}B transfectants compared with 1.47 and 1.45, respectively, for vector control transfectants). Expressions of Cyclin B1, Cyclin D1, and HIAP were down-regulated by the inhibition of NF-{kappa}B. These results suggest that transcription of NF-{kappa}B and a group of NF-{kappa}B target genes are involved in radioresistance in FIR-treated tumor cells with inactive TP53.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor radioresistance remains a critical obstacle in clinical radiotherapy or combined radiochemotherapy. TP534 -related signaling pathways have been well demonstrated in radiation-induced responses in cells with a functional TP53 (1 , 2) . However, ~50% of human tumors contain a mutant or inactivated TP53; and the TP53 protein of tumor cells, especially cervical cancer cells infected with HPV, may be inhibited by HPV E6 oncoproteins (3) . Radiation responses of these HPV-positive cervical cancers need to be elucidated. Radiation-induced stress responses are shown to be mediated via TP53-independent pathways (4 , 5) , and radiosensitivity and DNA repair ability are not directly related to TP53 activity (6 , 7) . These results suggest that, in addition to TP53, alternative signaling pathways play a critical role in response to radiation-induced stress and radioresistance. Radiation response in TP53-inactive cervical tumor cells infected with HPV provides an opportunity for identifying different signaling events involved in tumor radioresistance.

To investigate clinically observed radioresistance, regimens of FIR in vitro have been used to determine molecular mechanisms underlying radioresistance. Although different responses have been reported in FIR-treated cells, significant resistance to radiation and/or chemotherapeutic agents has been observed in several tumor or transformed cells (8 , 9) . FIR reduced estrogen receptor expression and increased transforming growth factor {alpha} expression in MCF-7 breast cancer cells, but little radioresistance was observed (10) . In contrast, fractionated X-ray-irradiation resulted in distinctive resistance to chemotherapeutic agents in breast tumor cell lines (11) . In addition, we have reported that a radioresistant phenotype (DMF, 1.60) developed in breast cancer MCF-7 cells (with a wild-type TP53) after FIR (12) . Studies are needed to see whether FIR could also induce a radioresistant phenotype in tumor cells infected with HPV that would result in an inactive TP53.

NF-{kappa}B is a well-defined radiation-responsive transcription factor (13 , 14) . IR-induced NF-{kappa}B activates IKK, the protein kinase that phosphorylates I{kappa}B-{alpha} at Ser-32 and Ser-36 (15) . Inhibition of I{kappa}B-{alpha} phosphorylation at serine and tyrosine sites sensitizes human glioma cells to DNA damaging agents (16) . Aberrant regulation of NF-{kappa}B increased cell sensitivity to the cytotoxic effects of TNF-induced or radiation-induced apoptosis (17) and increased the radiosensitivity of ataxia telangiectasia cells (18) . Also, modulation of the activity of NF-{kappa}B as a target gene involved in radiosensitization has increased cell sensitivity in several tumor cell lines (19 , 20) , and down-regulation of NF-{kappa}B is probably required for TP53-dependent apoptosis (21) . In addition, blocking radiation-induced NF-{kappa}B increased the apoptotic response and decreased the growth and clonogenic survival of colorectal cancer cells (22) . However, the inhibition of NF-{kappa}B in prostate cancer cells and HD-MyZ Hodgkin’s lymphoma cells did not affect radiosensitivity (23) , nor was cell sensitivity to the cytotoxic effects of TNF and chemotherapeutic agents increased (24) . Because TP53 and NF-{kappa}B can cross-talk in signaling different stress conditions (25) , a study is needed to differentiate the effect of NF-{kappa}B activity on radiosensitivity from the effect of TP53 activation on radiosensitivity. In fact, understanding signaling pathways that are responsive to NF-{kappa}B activation when TP53 is inhibited is essential for identifying molecular events causing radioresistance in TP53-inactive tumor cells.

Many stress-responsive genes are up-regulated in gene expression profiles obtained from radiation-treated cells (26) . A large group of radiation-responsive genes are controlled by both TP53 and NF-{kappa}B. Two stress-related proteins, metallothionein and Ku-autoantigen, activated in cells with a radioresistant phenotype are responsive to NF-{kappa}B regulation (27 , 28) . Radiation also induces other NF-{kappa}B target genes such as IL-1ß, IL-6, and TNF-{alpha} (29) , intercellular adhesion molecule-1 (ICAM-1; Ref. 30 ), manganese superoxide dismutase (31) , galectin (32) , E-selectin (33) , and {gamma}-glutamylcysteine synthetase ({gamma}-GCS, the rate-limiting enzyme of glutathione synthesis involved in radioprotection). From a gene expression profile of breast cancer MCF-7 cells treated with FIR (12) , up-regulated Cyclin B1 was found to be one of the critical elements responsible for FIR-induced radioresistance. These FIR-responsive signaling elements should be studied in relation to the radiation response of TP53-inactive tumor cells.

In this study, we measured NF-{kappa}B transactivation and a group of genes up-regulated after FIR in a human keratinocyte HK18 cell line that is TP53-inactive because of expression of the HPV18 E6 gene (35 , 36) . Results obtained with this cell model are informative for the study of the radiation response in human cervical cancers that are positive for the HPV infection that causes a dysfunctional TP53. Our results suggest that NF-{kappa}B activation and a group of genes involved in cell cycle control and apoptosis are at least partially responsible for radioresistance induced by FIR in TP53-inactive keratinocytes.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Keratinocytes and FIR Treatments.
The human keratinocyte cell line (HK18) established by cotransfection with HVP18 genome and v-fos (35) at passage number 98 was maintained in 3:1 KGM:DMEM (2.5% fetal bovine serum and 5% CO2) as described previously (36) . This low-fetal-bovine-serum medium was optimized to grow the HK18 keratinocyte without inducing differentiation or growth arrest. Radiation-resistant keratinocytes (HK18-IR) were derived by FIR treatment of HK18 cells with a dose of 60 Gy (2 Gy/fraction, twice a week), delivered at room temperature at 46 cGy/min with a Theratron-80 S/N 140 Co-60 unit (Atomic Energy of Canada, Limited). HK18 cells, used as controls, were treated with the same procedure, except they were sham irradiated. Both FIR-treated and sham-FIR-treated control cells were passaged every 7 days before the eighth irradiation, and then were passaged every 10 days after the eighth irradiation. The number of cells plated was less for control HK18 cells than for HK18-IR cells to have the same number of passages for both cell lines.

Experiments were performed within seven passages after termination of FIR. For clonogenic assays, cells were plated into 60-mm dishes and were exposed to a range of doses. After irradiation, cells were cultured for 14 days, colonies having more than 50 cells were counted as surviving colonies, and the number of colonies were normalized to the number observed for unirradiated cells. To determine IR-induced growth delay, 2.5 x 104 cells were plated into 12-well cell culture flasks, incubated for 24 h, and then irradiated. Cell growth was monitored by counting cell numbers at different time intervals after a single dose of 5 Gy.

Flow Cytometry.
Both HK18 and HK18-IR cells were cultured in 3:1 KGM:DMEM, irradiated at 80–90% confluence with 5 Gy, and collected at different time intervals after radiation. Cells were fixed in 85% ethanol and stained with PI. The PI-stained nuclear DNA content, which indicated the distribution of cells in G1, S phase, and G2, was determined with a Becton Dickinson flow cytometer.

DNA Fragmentation Assay.
The HK18 and HK18-IR cells that were grown in KGM-DMEM were sham-irradiated or irradiated with a single dose of 5 Gy. Then, the cells were collected by trypsinization 24 h or 48 h after irradiation. Genomic DNA was prepared immediately after cell collection, and DNA fragments in unirradiated and irradiated cells were detected by electrophoresis.

Nuclear Protein Analysis.
Nuclear proteins were prepared from HK18 and HK18-IR cells (5 x 107) with the previously described method (36) . Briefly, cell lysates were centrifuged at 4°C for 1 min, and the pellets were washed once with 0.3–0.5 ml of washing buffer (50 mM KCl, 25 mM HEPES, 2 mM PMSF, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 100 µM DTT) and then once by centrifugation again at 4°C for 1 min. The cell nuclei were then resuspended in 50–100 ml of extraction buffer (500 mM KCl, 75 mM HEPES, 10% glycerol, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µM DTT) and centrifuged at 4°C for 5 min. The supernatants were saved as nuclear extracts. Three µg of nuclear protein were reacted with radioactive-labeled TP53 and NF-{kappa}B oligonucleotides (Promega Co. Madison, WI), and the DNA-protein complexes were resolved in a 6% nondenaturing acrylamide gel by electrophoresing for 1.5–2 h at room temperature.

Reporter Transfection and Luciferase Assay.
Cells grown in 12-well plates were cotransfected with 0.3 µg of luciferase transcription factor reporters and 0.2 µg of ß-galactosidase reporters. NF-{kappa}B luciferase reporter transfection was the same as described previously (12) . Cells were transfected for 6 h, then sham-irradiated or irradiated with 5 Gy immediately after transfection. Luciferase activity was measured at different times after radiation. For normalization of luciferase expression, an aliquot of the same cell lysate was saved for measurement of B-galactosidase activity.

DNA Microarray Analysis.
To determine the effector genes regulated by NF-{kappa}B transactivation, DNA microarray analysis with 588 known human genes,5 including signaling elements involved in stress response and cell proliferation, was performed with paired comparisons of HK18-IR and HK18 control cells. The DNA microarray analysis was performed with the cells at, or within seven passages after, FIR when the radioresistant phenotype was observed (Fig. 1, A and B)Citation . Total RNA was extracted from the cells using TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD). After confirmation of the integrity of RNA on an agarose gel, RNA was digested by RNase-free DNase I for 20 min, extracted using phenol-chloroform, and then precipitated using 2.5 volumes ethanol. Polyadenylate+RNA was isolated using the Oligotex RNA kit (Qiagen Inc., Valencia, CA). Procedures for radioactive labeling and hybridization with the gene filter5 have been described previously (12 , 37) . After hybridization, array filters were stringently washed four times before being exposed to X-ray film overnight at -80°C. The filters were also exposed to a Phosphor Screen overnight and scanned using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and signals of paired genes in control HK18 and HK18-IR cells were quantified by ImageQuant software. Comparisons were made between the two cell types before irradiation (basal levels) or at 24 h after irradiation with 5 Gy.



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  		Fig. 1. Radioresistance induced in HK18 cells by FIR. A, clonogenic survival. The human keratinocyte cell line (HK18) cultured in KGM-DMEM was irradiated with fractionated doses (FIR, 2 Gy/fraction, twice a week, delivered at 46 cGy/min). Both irradiated (HK18-IR) and sham-irradiated control (HK18) cells were maintained in complete medium, and clonogenic survival was measured within six to eight passages after FIR. Cells plated in 60-mm dishes were irradiated with a range of doses, and colonies formed after incubation for 18 days were counted to calculate the survival fractions. The plating efficiencies of HK18-IR cells (0.18) and HK18 cells (0.17) were used for normalization of survival fractions; each datum point, the mean and SD of three experiments. B, growth delay of HK18-IR cells induced by 5 Gy. The parental HK18 and HK18-IR cells were plated in 12-well plates (2.5 x 104 cells/well) 24 h before irradiation. Cell numbers were counted at different times after 5-Gy irradiation. Cell multiplication of HK18 and HK18-IR cells without 5-Gy irradiation is also shown and used as controls. Data represent the average of four experiments.

 
Establishment of Mutant I{kappa}B Transfectants.
HK18-IR cells (passage 7 after FIR) were maintained in 3:1 KGM:DMEM. Stable transfectants were obtained using a previously described method (38) with Lipofect AMINE reagent (Life Technologies, Inc.). Briefly, 5 x 106 HK18-IR cells, grown in 100-mm cell culture dishes, were transfected with 15 µg of mutant I{kappa}B plasmid controlled by a cytomegalovirus promoter (provided by Dr. Nancy Rice at National Cancer Institute, NIH, Frederick, MD), 2 µg of hygromycin marker pCEP4, and 40 µg Lipofect AMINE in 6 ml of serum-reduced OPTI-EMEM (Life Technologies, Inc.). pcDNA3 in the place of mutant I{kappa}B was transfected as vector control. Cells were transfected for 72 h, trypsinized, and cultured in the selecting medium with 50 µg/ml hygromycin B for 14–20 days. The selected mutant I{kappa}B and vector control clones were pooled and passaged with 10 µg/ml hygromycin B. The transfected cells were cultured for at least two passages in hygromycin-B-free medium before experiments.

RNase Protection Assay and RT-PCR.
RNA was prepared by TRIzol Reagent (Life Technologies, Inc. Co., Grand Island, NY). Expression of cyclin kinase genes in HK18 and HK18-IR cells was analyzed by RNase protection assay using hCC-2 (human cell cycle) multiprobe sets (Promega). DNA probe sets were radioactive-labeled with [32P]UTP and hybridized overnight to 2 µg of total RNA that was isolated from cells at different times after irradiation. RNA preparations were treated with RNase-free DNase I before RT-PCR was performed. cDNA was synthesized in a final reaction volume of 20 µl containing 1 µg total RNA, 100 pmol of random hexadeoxynucleotides (Promega), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM DTT, dNTPs (500 µM each), 20 units of RNase inhibitor (rRNasin; Promega) and 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies, Inc.). The mixture was kept at room temperature for 10 min and then incubated at 42°C for 60 min. RNA samples from each cell line without reverse transcriptase were used in duplicate reactions as negative controls. For PCR reactions, 5 µl of cDNA products were mixed with PCR reaction buffer containing 5.0 mM MgCl2, 50 µM each dHTP, 2.0 units Taq DNA polymerase (Promega) and 0.1 µM each cDNA primer listed in Table 1Citation .


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  		Table 1 PCR primers

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Radioresistance in HK18 Cells after FIR.
Clonogenic survival after irradiation was determined for parental HK18 (sham FIR-treated) and HK18-IR (FIR-treated) keratinocytes. HK18-IR derived by FIR showed decreased radiosensitivity compared with the level of sham FIR-treated control HK18 cells (Fig. 1A)Citation . DMFs were 1.47 and 1.51, respectively at 10% and 1% of isosurvival levels for HK18-IR cells. Also, cell growth delay after 5-Gy irradiation was less for HK18-IR cells than for control HK18 cells. For ~5-fold increase in cell number, the delay was ~55 h for HK18-IR compared with ~83 h for control HK18 cells (Fig. 1B)Citation .

The difference in response to radiation was studied further by cell cycle analysis using flow cytometry. Table 2Citation shows that, in the control HK18 cells, the percentage of cells in G2 phase was doubled at 24 h and 48 h after 5 Gy irradiation. In contrast, in HK18-IR, the percentage in G2 was reduced almost 2-fold at 2 h and 24 h after 5 Gy. Also, HR18-IR cells without irradiation had relatively more cells in G2 compared with those in HK18 cells. There was no change in percentage of cells in S phase for HK18 cells, with or without irradiation; however, HK18-IR cells appeared to have more cells in S phase after irradiation. These results, together with the shortened growth delay observed in HK18-IR cells (Fig. 1B)Citation , suggest, but certainly do not prove, that radioresistance after FIR is associated with a reduced G2 delay.


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  		Table 2 Cell cycle distribution of HK18 and HK18-IR cells after 5-Gy irradiationa

 
Decreased Sensitivity of HK18-IR Cells to IR-induced Apoptosis.
We used flow cytometry to detect apoptotic cells (Fig. 2)Citation . No apoptotic cells were detected in HK18 and HK18-IR cells without radiation (Fig. 2, A and C)Citation . In contrast, after 5 Gy, a large portion of HK18 cells had DNA fragments (Fig. 2B)Citation compared with none of the HK18-IR cells having DNA fragments (Fig. 2D)Citation . These results were confirmed with DNA gel electrophoresis; i.e., laddering of genomic DNA was detected in HK18 cells but not in HK18-IR cells at 24 h and 48 h after 5 Gy (Fig. 2E)Citation .



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  		Fig. 2. HK18-IR cells were resistant to radiation-induced apoptosis. A–D, apoptotic cells detected by flow cytometry. HK18 cells without (A) and with (B) 5-Gy irradiation, and HK18-IR cells without (C) and with (D) 5-Gy irradiation were collected (5 x 104 cells) 24 h after radiation. Cells were fixed in 85% ethanol, stained with PI, and analyzed with a Becton Dickinson flow cytometer. Arrows, cells with less than 2N of DNA content, illustrate apoptotic cells in the irradiated HK18 cells (B). Cells with less than 2N DNA were absent in the irradiated HK18-IR cells (compare B and D). E, DNA fragments analyzed by gel electrophoresis. HK18-IR (FIR, +) and HK18 (FIR, -) cells grown in complete medium with or without 5-Gy irradiation were collected 24 h or 48 h after irradiation by trypsinization. DNA was prepared, and DNA fragments were detected by electrophoresis. Data represent one of three gels.

 
Activation of NF-{kappa}B in HK18-IR Cells.
We have reported that NF-{kappa}B is activated in human breast cancer MCF-7 cells treated with FIR (12) . To investigate the activity of NF-{kappa}B in TP53-inactive keratinocytes, we measured DNA-binding activity of TP53 and NF-{kappa}B (Fig. 3, A and B)Citation . Gel-shifting analysis showed that, in agreement with reported results of TP53 in HK18 cells that express the HPV18 E6/E7 gene (35 , 36) , DNA-binding activity of TP53 was absent in parental HK18 cells. Also, no activation was observed in HK18-IR cells, and irradiation with 5 Gy did not activate TP53 in either the parental HK18 or the HK18-IR cells (Fig. 3A)Citation . These results indicate that TP53, inhibited by HPV18 E6, is not activated by radiation. In contrast, the basal DNA-binding activity of NF-{kappa}B was elevated in HK18-IR cells relative to HK18 cells, and was activated further by 5 Gy in both HK18 and HK18-IR cells (Fig. 3BCitation , Lanes 6–10 and 12–14). In addition, the basal and radiation-induced NF-{kappa}B-dependent reporter-gene expression were activated more in HK18-IR cells than in HK18 cells (Fig. 3C)Citation .



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  		Fig. 3. NF-{kappa}B, but not TP53, was activated in HK18-IR cells. In all cases, "0" represents unirradiated cells. A and B, gel-shifting analysis of TP53 and NF-{kappa}B binding. Parental HK18 cells and HK18-IR cells were irradiated with 5 Gy, and nuclear proteins were extracted from cells at indicated times after irradiation. Gel-shifting analysis was performed with 3 µg of nuclear proteins and radioactive-labeled oligonucleotides of TP53 (A) or NF-{kappa}B (B). Lanes 1–4, nuclear extracts from HeLa cells used as a positive control (N, no nuclear protein; P, 32P-labeled oligo + nuclear protein; C, competitive control with cold oligo + nuclear protein; O, non-competitive control + nuclear protein). Lower panels (A and B), the estimated DNA binding activity. Data represent one of three gels quantified by densitometry and normalized to the value for the control (unirradiated HK18) cells at zero time (Lane 5). C, basal NF-{kappa}B reporter transcription and transcription (at 24 h after 5 Gy-irradiation) were increased in HK18-IR cells. Luciferase reporter of NF-{kappa}B was cotransfected with B-galactosidase reporter into parental HK18 and HK18-IR cells as described in "Materials and Methods." Cells were irradiated with 5 Gy immediately after transfection, and luciferase activity was measured at indicated times after irradiation. Data represent mean and SD from three experiments.

 
Candidate Genes Responsive to NF-{kappa}B Activation in HK18-IR Cells.
To determine what genes were altered in expression because of NF-{kappa}B activation in HK18-IR cells, gene expression profiles were obtained using DNA microarray analyses that included 588 human genes containing many elements involved in cell cycle control and apoptosis (12) . Table 3Citation shows genes that were matched in HK18-IR profiles compared with those matched in HK18 cells, both for basal levels before irradiation and for levels 24 h after 5 Gy. Note that the 2- to 5-fold up-regulation of the 25 genes in the HK18-IR cells relative to HK18 cells was similar when comparisons were made for basal levels or for levels after irradiation. The basal levels of several genes were higher for HK18-IR cells than for HK18 cells when the levels after irradiation were the same or lower for HK18-IR cells than for HK18 cells.


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  		Table 3 Genes up-regulated in HK18-IR cells in basal and 24 h after 5-Gy irradiation compared with parental HK18 cells

 
By analyzing from database searches the promoter regions of the up-regulated genes and their functional relationships to NF-{kappa}B transactivation, six genes from these profiles were selected as candidate genes responsive to NF-{kappa}B activation: Cyclin B1, Cyclin D1, BAG-1, HIAP, TTF, and fibronectin (Table 3)Citation . This selection was somewhat arbitrary because other genes in this list are also responsive to NF-{kappa}B activation. Cyclin B1 was selected because its up-regulation was found to be particularly responsible for the radioresistance of breast cancer MCF-7-IR cells that were derived from parental MCF-7 cells treated with FIR (12) . Also, the distribution in the cell cycle was altered in both irradiated MCF-7-IR cells and irradiated HK18-IR cells. These results suggest that activation of cell cycle elements, such as Cyclin B1 and Cyclin D1, by NF-{kappa}B may be responsible for the altered cell progression and radioresistance observed in FIR-treated cells. In addition, BAG-1 and HIAP have been identified with signaling antiapoptotic pathways (39, 40, 41) . Thus, our expression profiles have shown that a group of NF-{kappa}B target-signaling elements related to cell cycle control and apoptosis are activated in HK18-IR cells that have an inactive TP-53. Few genes, MP-13 (GenBank no. D26512), Caspase 2 (GenBank no. U13021), death-associated protein 3 (GenBank no. X83544), IGF-BF6 (GenBank no. M31145), insulin-like growth factor binding protein 2 (GenBank no. A09809), were found to have their basal levels down-regulated in HK18-IR cells compared with those in HK18 cells (2- to 5-fold lower than control HK18 cells).

Confirming the Elevated Expression of NF-{kappa}B-responsive Genes in HK18-IR Cells.
RT-PCR confirmed the increase in basal levels in HK18-IR cells relative to those in HK18 cells (Table 3)Citation for Cyclin B1, Cyclin D1, and two other up-regulated genes, HIAP and BAG-1 (Fig. 4)Citation . Also, the relative increase in the expression of these cell cycle elements in HK18-IR cells relative to HK18 cells was confirmed by an RNase protection assay (Fig. 5)Citation . Basal levels of Cyclin D1 and Cyclin B1 increased in HK18-IR cells, whereas the basal level of Cyclin D2 decreased. However, Cyclin B1, Cyclin D1, and Cyclin D2 were all induced by 5 Gy in both HK18 cells and HK18-IR cells (Fig. 5)Citation .



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  		Fig. 4. Basal expression of NF-{kappa}B target genes in HK18 cells and HK18-IR cells measured by RT-PCR. The basal expression of four NF-{kappa}B responsive genes up-regulated as detected by the DNA microarray analysis (Table 3)Citation was quantified by RT-PCR. Gene-specific primers were synthesized from the GenBank sequence database as described in "Materials and Methods" (Table 1)Citation . RT-PCR was performed with 1.0-ml cDNA products of parental HK18 (FIR, -) cells or HK18-IR (FIR, +) cells. GAPDH, included as control.

 


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  		Fig. 5. Expression of NF-{kappa}B target genes from HK18 and HK18-IR cells, measured by RNase protection. Expression of cyclin kinase genes [in HK18 (FIR, -) and HK18-IR (FIR, +) cells after 5-Gy irradiation] was analyzed by an RNase protection assay using human cell cycle multiprobe sets. DNA probe sets were radioactive-labeled with [32P]UTP and were hybridized overnight to 2 µg of total RNA isolated from cells at different times after irradiation. Time "0", without irradiation; mk, RNA marker. Both L32 and GAPDH were included as controls. Lower panel, relative levels of Cyclin B1, Cyclin D1, and Cyclin D2 measured in areas enclosed in the rectangles in upper panel. Data represent one of three experiments.

 
Radioresistance of HK18-IR Was Reduced by Transfecting Mutant I{kappa}B to Inhibit Expression of NF-{kappa}B.
HK18-IR cells were transfected with a mutant I{kappa}B (HK18-IR+mI{kappa}B cells) that has a dominant-negative phosphorylation domain that inhibits NF-{kappa}B/I{kappa}B dissociation (42 , 43) . Basal NF-{kappa}B activity was reduced ~5-fold in the HK18-IR+mI{kappa}B cells, i.e., to about the same level as in the HK18-IR vector control cells (Fig. 6A)Citation . Radiation-induced NF-{kappa}B transactivation also was abolished in HK18-IR+mI{kappa}B cells at 24 h after 5-Gy irradiation (Fig. 6A)Citation . There were, however, no significant differences in cell growth and plating efficiencies between these cell lines (data not shown).



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  		Fig. 6. A, inhibition of NF-{kappa}B activity by mutant mI{kappa}B transfection. mI{kappa}B plasmids and control vectors were transfected into HK18-IR cells, and stable transfectants were selected. NF-{kappa}B activities of the parental control (HK18), the transfectant of the vector control (HK18-IR+V), and the mI{kappa}B transfectant (HK18-IR+mI{kappa}B) were measured by cotransfection of NF-{kappa}B luciferase reporter and ß-galactosidase reporter. Cells were either sham-irradiated (0) or were irradiated with 5 Gy immediately after luciferase reporter transfection, and luciferase activity was measured 24 h later. Luciferase activity was normalized to ß-galactosidase activity, and results represent the means and SD from three experiments (**, P < 0.01). B, inhibition of radioresistance in mI{kappa}B transfectants. HK18, HK18-IR+V, and HK18-IR+mI{kappa}B cells plated with different cell numbers in 60-mm cell culture dishes were exposed to IR (0–12 Gy). After irradiation, cells were cultured for 14 days, and colonies with more than 50 cells were counted to calculate the clonogenic survival. The plating efficiencies of 0.17 for control HK18 cells, 0.18 for HK18-IR+V cells, and 0.12 for HK18-IR+mI{kappa} B cells were used for calculating normalized survival fractions for each cell population. The data represent means and SD from three experiments.

 
Next, we determined the effect of NF-{kappa}B inhibition on the radioresistance of HK18-IR cells. In Fig. 6BCitation , the radiation survival of the HK18-IR cells, transfected with mutant I{kappa}B (HK18-IR+mI{kappa}B cells), was reduced and approached the survival of the HK18 cells. DMFs of HK18-IR+mI{kappa}B cells were 1.18 and 1.10, respectively, at 10% and 1% of isosurvival compared with 1.47 and 1.45 for vector control transfectants.

Inhibition of Expression of NF-{kappa}B-responsive Genes in HK18-IR+mI{kappa}B Cells.
We then determined the effect of regulating NF-{kappa}B activity on expression of Cyclin B1, Cyclin D1, HIAP, and BAG-1 at 24 h after 5-Gy irradiation. Expression levels were measured in HK18-IR cells and in HR18-IR cells transfected with mutant I{kappa}B (HK18-IR+mI{kappa}B cells; Fig. 7Citation ). Reducing the transcriptional activity of NF-{kappa}B with mutant I{kappa}B reduced the expression levels of Cyclin B1, Cyclin D1, and HIAP by 4- to 5-fold, but caused less than a 2-fold reduction in expression of BAG-1. As expected, no activation of any of these genes was observed in the mutant I{kappa}B transfectants after 5-Gy irradiation (data not shown). These results indicate that at least one group of signaling elements linked to NF-{kappa}B regulation are activated in the radioresistant HK18-IR cells, and that their activation may play a role in radioresistance via alteration of cell cycle progression and the apoptotic response.



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  		Fig. 7. mI{kappa}B transfection down-regulated NF-{kappa}B target genes. Expression of four NF-{kappa}B target genes up-regulated in HK18-IR cells (see DNA microarray analysis in Table 3Citation ) was measured in vector control cells (HK18-IR+V: mI{kappa}B, -) and in HK18-IR cells transfected with mutant I{kappa}B (HK18-IR: mI{kappa}B, +). RT-PCR was performed with specific gene primers listed in Table 1Citation using total RNA isolated from (HK18-IR: mI{kappa}B, +) cells and vector control cells (HK18-IR+V: mI{kappa}B, -) 24 h after 5-Gy irradiation. Expression levels were measured by densitometric scanner analysis and normalized with GAPDH (right panel, data represent one of three experiments).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Radiation response in HPV-positive tumors that causes a dysfunctional TP53 (such as cervical cancers positive for HPV) is an essential issue in anticancer treatments with radiation. The inactivation of TP53 by HPV-coded E6 protein is mediated by ubiquitin-mediated degradation and, thus, is different from the mutant TP53 proteins found in TP53-mutant tumor cells. The molecular mechanisms underlying the radiation response in tumor cells with HPV-inactivated TP53 needs to be elucidated. In the present study, we provide the first experimental evidence that transcription factor NF-{kappa}B and a subset of NF-{kappa}B-regulated cell cycle genes and antiapoptosis response genes are activated in chronically irradiated human keratinocytes (HK18) that are TP53 inactive (Table 3Citation and Figs. 3Citation 4Citation 5Citation ) because of infection of HPV18. After chronic irradiation delivered in 30 fractions of 2 Gy each (FIR), the derived HK18-IR cells treated with a single dose of irradiation had a shortened growth delay and increased clonogenic survival (Fig. 1)Citation and were resistant to apoptosis (Fig. 2)Citation . Abolishing up-regulation of NF-{kappa}B in HK18-IR cells by transfecting mutant I{kappa}B inhibited expression of Cyclin B1, Cyclin D1, and HIAP (Fig. 7)Citation and reduced radioresistance (Fig. 6)Citation . These results suggest that NF-{kappa}B transactivation regulates the expression of genes that are responsible for the radioresistance observed in TP53-inactive keratinocytes. The genes up- regulated after FIR could be activated via TP53-dependent or -independent pathways because both a radioresistant phenotype and an up-regulation of NF-{kappa}B were observed in TP53 wild-type MCF-7 cells (12) and in TP53-inactive HK18 cells in the present study.

To elucidate the mechanisms involved in radioresistance, we need to further define the specific transcription factors and downstream target genes responsible for radioresistance. Presumably, the signal elements regulated by the transcription factors, instead of the transcription factors themselves, provide specific molecular targets that determine radiosensitivity. In the present study, we have identified from the DNA binding of NF-{kappa}B and reporter transfection analysis that transcriptional activity of NF-{kappa}B is apparently involved in the radioresistance of HK18-IR cells (Fig. 6)Citation . From the list of 25 genes up-regulated in the HK18-IR cells, we have identified 6 genes as putatively under NF-{kappa}B regulation (Table 3)Citation . Of these six genes, Cyclin B1, Cyclin D1, and HIAP were down-regulated in HK18-IR cells after inhibiting transcription of NF-{kappa}B by the transfecting of a dominant–negative mutant I{kappa}B (Fig. 7)Citation ; three genes, BAG-1, TTF, and fibronectin, were not down-regulated significantly (Fig. 7Citation and data not shown). Because the down-regulation of these three genes correlated with a large decrease in the survival of HK18-IR cells after irradiation, we might conclude that expression of any or all of the three genes, i.e., Cyclin B1, Cyclin D1, and HIAP, play a major role in the radioresistant phenotype. Abrogation of NF-{kappa}B activity did not significantly down-regulate BAG-1, TTF, or fibronectin. Therefore, either these three genes do not play a role in radioresistance or they are regulated by other transcription factors.

Now, studies using antisense or dominant-negative technology were performed for each of the three genes in radiation survival. When this was done for Cyclin B1 and p21 in radioresistant MCF-7-IR cells, radioresistance that was quantified by increased clonogenic survival and reduced growth delay correlated with the up-regulation of Cyclin B1; and when expression of Cyclin B1 was inhibited in the MCF-7-IR cells, radioresistance was reduced greatly (12) . An important question is whether the same relationship between expression of Cyclin B1 and radioresistance will be seen in radioresistant HK18-IR cells, and if so, does up-regulation of any of the other two genes also contribute to radioresistance. Finally, because only a portion of the radioresistance appeared to be attributed to up-regulation of NF-{kappa}B (Fig. 6)Citation , antisense or dominant-negative technology is also needed to explore the role that any of the other 25 up-regulated genes controlled by transcription factors other than NF-{kappa}B play in radioresistance. Quite likely, the few genes (0.8%), such as Cyclin D2 (Fig. 5)Citation that are down-regulated in HK18-IR cells, do not play a role in radioresistance.

In the present study and in the previous study with MCF-7 cells (12) , we have not related radioresistance to the magnitude of gene expression induced after a single dose of irradiation. However, the paired comparisons of HK18-IR and HK18 parental cells for basal expression levels before irradiation or for expression levels at 24 h after 5 Gy of radiation indicate that the amount of up-regulation is very similar for both end points. Kinetic studies of gene expression as a function of time after irradiation are needed to determine whether changes in gene expression after irradiation are important. Nevertheless, based on our previous study with MCF-7 cells (12) and the present study with HK18 cells, we conclude that basal levels of gene expression before irradiation can affect radiosensitivity. Studies are in progress to determine whether the up-regulation of NF-{kappa}B that is associated with radioresistance in the HR18-IR population was induced in the parental HR18 cells during FIR, or instead occurred in a FIR-selected radioresistant subpopulation that existed in the parental population.

The literature indicates that FIR of cultured mammalian cells induces different cellular responses, depending on tumor cell lines, the radiation regimen, and cell passages after FIR (8 , 44) . Human breast cancer MCF-7 cells exposed to FIR showed no change in growth rate, although estrogen receptor expression was reduced, and plating efficiencies were increased (10) . A 1.5-fold of radioresistance was induced in FIR-treated human T-cell leukemia cells with a 6-fold increase in expression of a multidrug resistance-associated protein (45) . We observed a radioresistant phenotype (DMF, 1.6–1.8 at 10% of isosurvival rate) in MCF-7 cells after FIR with 20 fractions (12) . The heterogeneity among cells surviving FIR as well as the heterogeneity in parental cell population could play a key role in FIR-induced radioresistance. Results reported by Russell et al. (8) and Li et al. (12) showed that the resistance was lost when the FIR-treated cells were growing in vitro without irradiation, but the resistant phenotype were found to be stable in several cloned cell lines. In the present study, we demonstrated that FIR induces a radioresistant phenotype in HK18 cells that have an inactive TP53 because of HPV18 transfection (3) . The radioresistant phenotype was also reversible in HK18-IR cells after 15 passages after FIR (data not shown). These results suggest that the radiosensitivity of individual clones is different, which can be the result of the heterogeneity of parental tumor population, or genes induced by FIR, or both. Genotyping analysis for individual clones isolated before and after FIR is under way.

Radioresistance was induced in either TP53-wild-type MCF-IR cells (12) or TP53 functionally knockout HK18-IR cells (present study). These results suggest a less relationship between TP53 activity and FIR-induced adaptive response. NF-{kappa}B, however, as a stress-responsive transcription factor, has been indicated to play a critical role in radiation response (46) . Up-regulation of NF-{kappa}B was observed after in vivo irradiation (29 , 47) and in both MCF-IR (12) and HK18-IR cells (Fig. 3)Citation . In addition, targeting the radiation-inducible transcription factors has tested tumor sensitization, and different results have been obtained. For example, expression of wild-type TP53 increased the apoptotic response shortly after irradiation but did not change clonogenic survival (4) . Adenovirus delivery of the TP53 gene increased ovarian tumor radiosensitivity (48) , whereas the inhibition of NF-{kappa}B by transfection with I{kappa}B did not affect radiosensitivity (23) . This report is in contrast to our present study, in which transfection of mutant I{kappa}B inhibited NF-{kappa}B activity and greatly reduced radiation survival of the radioresistant HK18-IR cells. In addition, induction of AP-1 components c-jun and c-fos, were found to have little effect on radiation-induced apoptosis in Jurkat T cells (49) . Thus, blocking the activities of radiation-inducible transcription factors seems to have different effects on radiosensitization probably because of the different functions of genes controlled by the individual transcription factors.

Recent results suggest that NF-{kappa}B and TP53 collaborate in regulating cell cycle checkpoints (50 , 51) . However, TP53 is inactive in the HK18 cells; therefore, downstream effects from the up-regulation of NF-{kappa}B are independent of TP53 (Fig. 3)Citation . In fact, our results (Figs. 1Citation and 3Citation ) suggest that NF-{kappa}B alone is able to influence cell cycle regulation (52 , 53) . The downstream effects of NF-{kappa}B activation, identified as up-regulation of Cyclin B1 and Cyclin D1 in the HK18-IR cells, are implicated in cell cycle checkpoints after irradiation. Up-regulation of Cyclin D1 might be expected to reduce the delay from G1 into S phase (54) . Although there was no obvious change in the distribution in the cell cycle after irradiation, other than a decrease in the accumulation of HK18-IR cells in G2 at 2 and 24 h after 5 Gy (compare with HK18 and HK18-IR cells in Table 2Citation ). In fact, the reduced accumulation of HK18-IR cells in G2 is consistent with the reduced growth delay for HK18-IR cells (Fig. 1)Citation and with the increase in levels of Cyclin B1 (Fig. 4Citation and 5Citation , and Table 3Citation ). A relationship between radiation-induced G2 delay and down-regulation of Cyclin B1 has been reported (55) . Up-regulation of Cyclin B1 has been indicated as a rate-limiting component for cells going from G2 to M phase (55) because a complex between Cyclin B1 and phosphorylated CDC2 occurs after irradiation (56) . Expression of Cyclin B1 decreased in HeLa cells irradiated in S phase (57) , whereas the amount of Cyclin B1 increased as the duration of G2 delay increased (58) . Furthermore, G2 delay decreased when Cyclin B1was induced using a controlled transgene promoter (55) . These results and our data showing a reduction in radiation-induced growth delay and an increase in the levels of Cyclin B1 in both MCF-7-IR cells (12) and HK18-IR cells (Figs. 1Citation , 4Citation , and 5Citation ) support the hypothesis that a certain level of Cyclin B1 is needed for irradiated cells to progress from G2 into mitosis. In addition, antisense Cyclin B1 reduced radioresistance of MCF-IR cells (12) . Furthermore, studies with computerized video-time-lapse of human bladder cells EJ30 delay in Dr. W. Dewey’s laboratory have shown that an increase in clonogenic survival is associated with a shortened division delay after irradiation.6 Experiments are in progress to confirm that the shortened G2 delay and the radioprotective response are causally associated with the increase in the level of NF-{kappa}B-regulated Cyclin B1.

Many radiation-inducible genes have been implicated in cells adapting to, or being protected from, radiation and chemotherapeutic agents (59) . Our present microarray results indicate that a group of stress-responsive genes were activated in HK18-IR cells and that about 25% of the up-regulated genes were identified as candidate genes responsive to NF-{kappa}B activation (Table 3)Citation . Although functions of many radiation-inducible genes are unknown, signal elements responsive to radiation are believed to induce radioresistance by cell cycle regulation, alterations in apoptosis, and changes in the ability to repair DNA damage (60 , 61) . Amundson et al. demonstrated that of 1238 human genes, 48 (3.87%) are inducible by a single dose of irradiation (62) . Similar alterations in gene expression were observed in array profiles from FIR-treated MCF-7 cells (3.9%; Ref. 12 ) and from FIR-treated HK18 cells (4.4%). These results suggest that changes in gene expression are similar in FIR-derived radioresistant cells and in cells exposed to a single dose of irradiation. However, as discussed above, increases in the basal levels of gene induction may be most relevant for radioresistance.

In the present study, we have focused on the increase in the basal activation of NF-{kappa}B that is associated with radioresistance after FIR. In Fig. 3Citation , the relative increase in radiation-induced NF-{kappa}B activity is much greater in HK18 cells (4-fold) than in HK18-IR cells (2-fold). However, we hypothesize that the elevated basal level of NF-{kappa}B activity in FIR-derived cells, rather than the induced activity, is responsible for at least part of the radioresistance phenotype. New results reported by Bradbury et al. (63) suggest that the inhibition of basal NF-{kappa}B is related to indomethacin-induced radiosensitization. Therefore, reduction of the basal NF-{kappa}B activity by mI{kappa}B transfection should inhibit NF-{kappa}B activity and increase radiosensitivity. However, transfection of mI{kappa}B into HK18-IR cells did not restore their radiosensitivity to the level of control HK18 cells (Fig. 6B)Citation . This may possibly result from stress-responsive transcription factors other than NF-{kappa}B (such as AP-1) and genes not regulated by NF-{kappa}B being involved in the signaling network for radioresistance. For example, antisense inhibition of the up-regulation of Cyclin B1 in MCF-IR cells after FIR did not totally abolish FIR-induced radioresistance (12) . Activation of NF-{kappa}B has also been shown to have an antiapoptotic function that is supposedly mediated through the induction of antiapoptotic genes. One of the antiapoptotic genes up-regulated in HK18-IR cells is HIAP (c-IAP2, MIHC) whose NF-{kappa}B-dependent transcription is dependent on two NF-{kappa}B sites bound by the NF-{kappa}B p50/p65 heterodimer (40) . The constitutive expression of natural caspase inhibitors is implicated in the resistance to apoptotic stimuli that directly target caspases (41) . HIAP is shown to inhibit specifically caspases-3 and -7 (64) . Also, BAG-1 that is up-regulated in HK18-IR cells is classified as an antiapoptotic gene (39) . Our results show that radioresistance in HK18-IR cells correlates well with levels of HIAP because the gene is activated in HK18-IR cells and is inhibited by mutant I{kappa}B (Fig. 6Citation and 7Citation ). However, as described above, other antiapoptotic genes activated by NF-{kappa}B and other transcription factors not examined in this study could also be involved in signaling resistance to radiation-induced apoptosis. Also, studies are needed to elucidate the molecular mechanisms underlying FIR-induced NF-{kappa}B activation. The function of signaling elements in the HK18-IR gene expression profiles involved in I{kappa}B phosphorylation and NF-{kappa}B nuclear translocation is worth being investigated further.

In conclusion, the present study demonstrates that FIR results in radioresistance in human keratinocytes that have TP53 inhibited because of expression of the HPV E6 gene. The FIR-derived radioresistant HK18 cells showed activation of NF-{kappa}B and an increase in expression of a group of genes. In particular, the up-regulation of three of the genes, Cyclin B1, Cyclin D1, and HIAP, were shown to be controlled by the activation of NF-{kappa}B. These results suggest that the activation of NF-{kappa}B and specific down-stream target genes form a signaling network for the radioresistance induced by FIR in TP53-inactive tumor cells infected with HPV.


   ACKNOWLEDGMENTS
 
We thank Dr. William Dewey at University of California-San Francisco for discussion of experiments and assistance in preparation of the manuscript; Dr. Richard Schlegel at Georgetown University, Washington, DC, for the kind gift of the HPV18 cell line; Dr. Nancy Rice at National Cancer Institute, NIH, Bethesda, MD, for providing mutant I{kappa}B construct; Dr. William McBride at University of California-Los Angeles for critical reading of the manuscript;, and Dr. Jeffrey Longmate at City of Hope National Medical Center for assistance in statistical analysis.


   FOOTNOTES
 
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 Supported in part by NIH Grant CA85344 (to B. H. S.) and by an intramural funding of Beckman Research Institute of City of Hope to J. J. L. Back

2 Present address: University of California-Davis, School of Medicine, Level 2, 1 Shields Avenue, Davis, CA 95616. Back

3 To whom requests for reprints should be addressed, at Halper South Building H115, 1500 Duarte Road, Duarte, CA 91010. Phone: (626) 301-8355; Fax: (626) 301-8892; E-mail: jjli{at}coh.org Back

4 The abbreviations used are: TP53, p53; DMF, dose-modifying factor; FIR, fractionated ionizing radiation; HPV, human papillomavirus; IR, ionizing radiation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; I{kappa}B, inhibitory {kappa}B; RT-PCR, reverse transcriptase-PCR; NF-{kappa}B, nuclear factor {kappa}B; TNF, tumor necrosis factor; KGM, keratinocyte growth medium; PI, propidium iodide. Back

5 Gene names and GenBank numbers are listed on Clontech web site: http://www.clontech.com. Back

6 W. Dewey, personal communication. Back

Received 8/23/01. Accepted 12/10/01.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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