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Molecular Cancer Research 3:345-353 (2005)
© 2005 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Enhanced G2-M Arrest by Nuclear Factor-{kappa}B-Dependent p21waf1/cip1 Induction

Shelly M. Wuerzberger-Davis1,3, Pei-Yun Chang2,3, Craig Berchtold3 and Shigeki Miyamoto1,2,3

1 Cancer Biology Program, 2 Molecular and Cellular Pharmacology, and 3 Department of Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin

Requests for reprints: Shigeki Miyamoto, Department of Pharmacology, University of Wisconsin, 301 Service Memorial Institute, 1300 University Avenue, Madison, WI 53706. Phone: 608-262-9281; Fax: 608-262-1257. E-mail: smiyamot{at}wisc.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) regulates cell survival pathways, but the molecular mechanisms involved are not completely understood. Here, we developed a NF-{kappa}B reporter cell system derived from CEM T leukemic cells to monitor the consequences of NF-{kappa}B activation following DNA damage insults. Cells that activated NF-{kappa}B in response to ionizing radiation or etoposide arrested in the G2-M phase for a prolonged time, which was followed by increased cell cycle reentry and survival. In contrast, those that failed to activate NF-{kappa}B underwent transient G2-M arrest and extensive cell death. Importantly, p21waf1/cip1 was induced in S-G2-M phases in a NF-{kappa}B-dependent manner, and RNA interference of this cell cycle regulator reduced the observed NF-{kappa}B-dependent phenotypes. Thus, cell cycle–coupled induction of p21waf1/cip1 by NF-{kappa}B represents a resistance mechanism in certain cancer cells.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The Rel/nuclear factor-{kappa}B (NF-{kappa}B) family of transcription factors regulates expression of genes critical for multiple biological processes, including immune responses, inflammatory reactions, and apoptosis (1, 2). In particular, many recent studies underscore the importance of NF-{kappa}B in the induction of genes involved in resistance to chemotherapeutic agents and radiation therapy (reviewed in ref. 3). These genes include antiapoptotic Bcl-2 family members and the cIAP family of caspase inhibitors, etc. (4-8). However, the repertoire of NF-{kappa}B-regulated genes that participate in cell survival regulation is incompletely understood.

Inactive NF-{kappa}B complexes exist in the cytoplasm in association with inhibitors, such as I{kappa}B{alpha}, and release from these inhibitors is critical for NF-{kappa}B to enter the nucleus and activate gene expression (1). Many signaling cascades that control NF-{kappa}B activation converge on an I{kappa}B kinase complex that is responsible for releasing NF-{kappa}B. Phosphorylation of I{kappa}B{alpha} on Ser32 and Ser36 leads to its ubiquitination and subsequent degradation by the 26S proteasome (9). Because of the sequential nature of this signaling pathway, there are many steps in which the activation of NF-{kappa}B can be inhibited. One strategy is to express a mutant form of I{kappa}B{alpha} (S32/36A-I{kappa}B{alpha} or so-called superrepressor I{kappa}B{alpha} mutant) that harbors mutations at the I{kappa}B kinase phosphorylation sites and consequently is not targeted for the degradation pathway to liberate NF-{kappa}B. Alternatively, although not as selective for the pathway as the above I{kappa}B{alpha} mutant, proteasome inhibitors have also been employed to prevent NF-{kappa}B pathways (10-13). Combined with knockout studies of the NF-{kappa}B family and I{kappa}B kinase components, these "loss of function" approaches were instrumental in determining the role of NF-{kappa}B as a key survival factor in both physiologic and pathologic settings (14-17). These approaches also helped to define NF-{kappa}B-regulated genes whose expression was lost on inhibition of NF-{kappa}B activation, thus correlating with its antiapoptotic activities.

However, the role of NF-{kappa}B in cell death regulation varies greatly depending on the cellular contexts and the stress signals used (18, 19). This disparity is presumably due to the differences in regulation of NF-{kappa}B target genes under different experimental settings. These discrepancies may, in part, stem from differences in both the percentage of a cell population among drug-exposed cells that is capable of activating NF-{kappa}B and the magnitude of activation within each stressed cell, both of which may vary greatly under different conditions. For example, when cells are treated with topoisomerase I inhibitors, such as camptothecin and its derivatives, only cells in the S phase seem to activate NF-{kappa}B efficiently (20, 21). Thus, any NF-{kappa}B-dependent effects will be initiated primarily from this cell population. Variations in the percentage of S-phase populations in drug-exposed cells will then introduce variations in NF-{kappa}B-dependent phenotypes. In other conditions, such as anticancer DNA-damaging agents, including ionizing radiation (IR) and the topoisomerase II inhibitor etoposide (VP-16), it is unknown whether NF-{kappa}B activation is regulated in a cell cycle–dependent manner or other undefined cellular contexts. Without a means to trace live NF-{kappa}B-activated cell populations, it is difficult to examine the populational variation of NF-{kappa}B activation and the consequences within those NF-{kappa}B-activated cells. Thus, there is a need to develop a cell-based NF-{kappa}B reporter system to evaluate the behavior of these distinct cell populations to directly link NF-{kappa}B activation to specific cell survival gene regulation and phenotypes.

To address cancer resistance mechanisms in relation to cell cycle regulation by NF-{kappa}B activation, we developed a "positive selection" strategy by engineering a NF-{kappa}B reporter cell system using a CEM T leukemic cell line. This system revealed a previously unrecognized G2-M role for NF-{kappa}B by inducing p21waf1/cip1 in S-G2-M cell cycle phases. p21waf1/cip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors involved in cell cycle and apoptosis regulation (22-24). This NF-{kappa}B-dependent regulation was not limited only to CEM cells because MDA-MB-231 breast cancer cells also showed similar phenotypes. Thus, our study suggests that the NF-{kappa}B-dependent p21waf1/cip1-G2-M arrest in part contributes to cancer resistance.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
NF-{kappa}B Activation by Genotoxic Agents in CEM Cells
Before developing our cell-based NF-{kappa}B reporter system, we determined the time course and dose response of NF-{kappa}B activation with multiple cell lines, including CEM cells, using distinct DNA-damaging agents. Cells treated with IR induced dose-dependent NF-{kappa}B activation that was saturated at ~10 Gy (Fig. 1A) and transient activation that peaked ~3 to 4 hours (Fig. 1B). Consistent with previous studies (25, 26), this activation did not require de novo protein synthesis and was associated with degradation of I{kappa}B{alpha} protein by 30 minutes, which was then followed by its resynthesis ~2 hours (Fig. 1B; others not shown). Treatment with DNA topoisomerase I or II inhibitors, such as camptothecin, topotecan, doxorubicin, and VP-16, also resulted in increased NF-{kappa}B DNA-binding activity (Fig. 1C) in a dose-dependent and similar transient activation manner (data not shown). Increased transcription of an NF-{kappa}B-dependent luciferase reporter gene (Fig. 1D) confirmed that NF-{kappa}B released by these DNA-damaging agents in CEM cells was transcriptionally competent.



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  		FIGURE 1. Double-strand break–inducing agents activate NF-{kappa}B in CEM human T cells. A. CEM cells were exposed to various doses of IR (1-40 Gy) and cells were collected 3 hours later. Total cell extracts were analyzed by EMSA using an Ig{kappa}-{kappa}B probe. Bottom, EMSA using an Oct-1 probe for a loading control. B. CEM cells were exposed to 10 Gy IR. Cell samples were terminated at indicated time points, and cell extracts were analyzed by EMSA as above (top) or Western blotting with I{kappa}B{alpha} antibody (bottom). C. CEM cells were treated with various genotoxic agents [10 µmol/L camptothecin (CPT), 20 µmol/L topotecan (TPT), 10 µmol/L VP-16 (VP16), 25 µmol/L doxorubicin, or 20 Gy IR] for 3 hours or TNF-{alpha} (10 ng/mL) for 20 minutes. NF-{kappa}B and Oct-1 binding activities were determined by EMSA as described above. D. CEM cells were transiently transfected with the 3x{kappa}B-Luc reporter plasmid. At 48 hours after transfection, cells were treated with TNF-{alpha}, camptothecin, doxorubicin, VP-16, or IR at the same doses as used in C for 6 hours. Cell extracts were analyzed for luciferase activity and standardized to total protein. Columns, mean of three independent experiments; bars, SD.

 
NF-{kappa}B Activation by Certain DNA-Damaging Agents Occurs in Different Cell Cycle Phases
To begin to monitor NF-{kappa}B functions at the single-cell level, we developed CEM{kappa}B, a CEM cell-based NF-{kappa}B reporter assay, by stably integrating the 3x{kappa}B-green fluorescent protein (GFP) reporter construct in a CEM T leukemic cell line. Multiple rounds of fluorescence-activated cell sorting (FACS) were used to isolate cell clones that displayed low basal GFP fluorescence with high-inducible GFP fluorescence following treatment with tumor necrosis factor-{alpha} (TNF-{alpha}; Fig. 2A, middle, and B, top; others not shown). This process ensured the isolation of cells that had stably integrated the {kappa}B-GFP reporter at inducible genomic loci. To ensure that the GFP-positive cells did indeed activate NF-{kappa}B and that NF-{kappa}B activation was low or undetectable in the GFP-negative cells, the two populations were also analyzed by electrophoretic mobility shift assay (EMSA) using the Ig{kappa}-{kappa}B site following TNF-{alpha} treatment. The GFP-positive cells displayed NF-{kappa}B-binding activity following TNF-{alpha} treatment, whereas GFP-negative cells displayed relatively little NF-{kappa}B-binding activity (Fig. 2C). Specific NF-{kappa}B-dependent induction of GFP was confirmed by its inhibition following expression of the superrepressor I{kappa}B{alpha} mutant protein (data not shown). Interestingly, the total number of GFP-positive cells after treatment with camptothecin was much less than those treated with TNF-{alpha} (Fig. 2A, bottom, and B, bottom). Moreover, cell sorting for GFP-positive and GFP-negative populations followed by propidium iodide (PI) staining showed that the GFP-positive populations following treatment with camptothecin were greatly enriched for only the S-phase cells, unlike TNF-{alpha}-stimulated cells that showed GFP induction in different cell cycle phases (Fig. 2D). These results in live cells showed that NF-{kappa}B activation by camptothecin was largely associated with the S phase, an observation consistent with previous biochemical studies (20, 21).



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  		FIGURE 2. NF-{kappa}B activation by camptothecin but not by IR and VP-16 is cell cycle dependent. A. CEM{kappa}B cells were treated with 10 ng/mL TNF-{alpha} or 10 µmol/L camptothecin for 24 hours. NF-{kappa}B activation was visualized under fluorescein-aided fluorescent microscopy (right) or phase-contrast microscopy (left). B. CEM{kappa}B cells were treated for 6 hours with 10 ng/mL TNF-{alpha} (top) or 10 µmol/L camptothecin (bottom). NF-{kappa}B activation was determined by measuring GFP fluorescence on a flow cytometer. C. CEM{kappa}B cells were treated with 10 ng/mL TNF-{alpha} for 24 hours. After treatment, GFP-positive cells were sorted from GFP-negative cells by flow cytometry. Total cell extracts were analyzed by EMSA using an Ig{kappa}-{kappa}B probe. CEM{kappa}B cells were treated for 6 hours with 10 ng/mL TNF-{alpha} (top) or 10 µmol/L camptothecin (bottom). NF-{kappa}B activation was determined by measuring GFP fluorescence on a flow cytometer. D. CEM{kappa}B cells were treated with 10 ng/mL TNF-{alpha} or 10 µmol/L camptothecin for 6 hours. After treatment, GFP-positive cells [GFP(+)] were sorted from GFP-negative cells [GFP(–)] by flow cytometry. Cell cycle profiles were examined by PI staining and analyzed with the CellQuest and ModFit software. E. CEM{kappa}B cells were treated with 10 µmol/L VP-16 for 6 hours and GFP-positive cells were sorted from GFP-negative cells by flow cytometry and analyzed as in D. F. CEM cells were pretreated with 25 µmol/L aphidicolin (Aph.) for 30 minutes (+ and –) followed by a 20-minute exposure to 10 ng/mL TNF-{alpha} or a 3-hour exposure to 10 µmol/L camptothecin, 10 µmol/L VP-16, 25 µmol/L doxorubicin, or 20 Gy IR. Cell extracts were analyzed for NF-{kappa}B (top) or Oct-1 (bottom) by EMSA.

 
Using this assay, we wanted to determine whether other DNA-damaging agents also activated NF-{kappa}B in specific phases of the cell cycle. In contrast to activation by camptothecin, which was efficiently prevented by the treatment with the DNA polymerase inhibitor aphidicolin (Fig. 2F, lane 4), NF-{kappa}B activation by none of the other DNA-damaging agents was sensitive to inhibition by this inhibitor. Thus, S-phase progression was not necessary to cause NF-{kappa}B activation by these other genotoxic agents. To directly determine whether NF-{kappa}B activation by IR and VP-16 was also cell cycle dependent, CEM{kappa}B cells were sorted for GFP-positive populations at 6 hours following treatment with these agents. Like TNF-{alpha}, but unlike camptothecin (Fig. 2D), the GFP-positive populations after VP-16 or IR exposure showed DNA contents representing each phase of the cell cycle (Fig. 2E; others not shown). Examining the DNA contents of total cell populations before and 6 hours after treatments showed that the differences seen between these GFP-positive populations were not due to overt differences in cell cycle progression within the assay period (see below).

CEM Cells Exhibit a NF-{kappa}B-Dependent G2-M Cell Cycle Arrest
Previous studies linking NF-{kappa}B activation with antiapoptotic activities relied primarily on either (a) increased apoptotic responses when NF-{kappa}B activation was prevented by the expression of a superrepressor I{kappa}B{alpha} mutant (20, 27, 28), knockout of p65 (14, 15), or knockout of I{kappa}B kinase components (16, 17) or (b) increased resistance to apoptosis when cells were made to overexpress members of the NF-{kappa}B family (29, 30). To our knowledge, there have not been studies that specifically examined the phenotypes of NF-{kappa}B-activated cell populations without prior manipulations of the NF-{kappa}B activation potentials. Thus, we treated CEM{kappa}B cells with IR and FACS sorted them based on the expression of GFP at different time points to examine the behavior of GFP-positive and GFP-negative populations. Although both populations accumulated efficiently at the G2-M cell cycle phase within 24 hours of exposure (Fig. 3A), high percentages of GFP-positive cells remained in this cell cycle phase for up to 72 hours with little apoptosis. By 96 hours, some of these GFP-positive cells had reentered the cell cycle as detected by the emergence of G1- and S-phase cells. In contrast, GFP-negative cells failed to remain in the G2-M cell cycle phase for a prolonged period and underwent nearly complete apoptosis within 96 hours. Importantly, these effects were not due to the expression of GFP per se, because GFP-positive cells following exposure to TNF-{alpha} did not display the similar phenotype (Fig. 4).



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  		FIGURE 3. NF-{kappa}B-activated cells undergo a prolonged G2-M cell cycle arrest followed by cell cycle reentry. A. CEM{kappa}B cells were exposed to 10 Gy IR and allowed to repair for the times indicated. At each time point, CEM{kappa}B GFP-positive cells were sorted from GFP-negative cells using the FACSVantage cell sorter. Cells were immediately fixed with 70% ethanol followed by cell cycle analysis by PI staining and modeling as in Fig. 2D. The cell cycle profiles for the total, GFP-positive, and GFP-negative cell populations at different time points are indicated. B. Different CEM-S32/36A clones were irradiated with a dose of 20 Gy for 3 hours and monitored for their ability to activate NF-{kappa}B. The corresponding I{kappa}B{alpha} protein levels were determined by Western blot analysis. Asterisk, exogenous superrepressor I{kappa}B{alpha}. C. CEM and CEM-S32/36A-23 cells were irradiated with a dose of 20 Gy and allowed to recover for the times indicated. Cell cycle profiles were determined as above.

 


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  		FIGURE 4. NF-{kappa}B-activated cells treated with TNF-{alpha} do not display a G2-M arrest. CEM{kappa}B cells were exposed to 10 ng/mL TNF-{alpha} and allowed to recover for the times indicated. At each time point, CEM{kappa}B GFP-positive cells were sorted from GFP-negative cells using the FACSVantage cell sorter. Cells were immediately fixed with 70% ethanol followed by cell cycle analysis by PI staining and modeling as in Fig. 2D. The cell cycle profiles for the total, GFP-positive, and GFP-negative cell populations at different time points are indicated.

 
To distinguish whether the G2-M arrest seen in GFP-positive cell populations was due to NF-{kappa}B activation or to some associated parallel events, we expressed the superrepressor I{kappa}B{alpha} mutant (S32/36A-I{kappa}B{alpha}) in CEM cells and isolated stable clones that failed to efficiently activate NF-{kappa}B when stimulated with IR and VP-16 (Fig. 3B; others not shown). Figure 3B shows NF-{kappa}B activity in three different clones isolated. CEM-S32/36A-23 displayed the greatest inhibition of NF-{kappa}B activity, so this clone was used in further experiments. When these cells were stimulated with IR, cell populations displaying prolonged G2-M arrest were markedly reduced, and correspondingly increased apoptosis was observed (Fig. 3C). These results indicated that the activation of NF-{kappa}B was at least in part necessary to maintain G2-M arrest and protect CEM cells from apoptosis.

p21waf1/cip1 Is Induced in a NF-{kappa}B-Dependent Manner following IR Exposure Primarily in S-G2 Cell Cycle Phases
NF-{kappa}B could arrest cells in G2-M by preventing cell death from this cell cycle phase by inducing antiapoptotic gene(s) or by inducing cell cycle inhibitor(s). We therefore screened the expression of several antiapoptotic genes and cell cycle regulators following exposure of CEM and CEM S32/36A cells to IR by RNase protection assay (RPA). We found that p21waf1/cip1, an inhibitor of cyclin-dependent kinases, was specifically induced in CEM cells but not in CEM-S32/36A cells (Fig. 5A). In contrast, the antiapoptotic Bcl-XL gene was not induced (Fig. 5A). Because p21waf1/cip1 is known for its induction by the p53 tumor suppressor in G1 to cause a G1 cell cycle arrest (23, 24), we wondered how NF-{kappa}B-dependent induction of p21waf1/cip1 was associated with the observed G2-M cell cycle arrest. This cyclin-dependent kinase inhibitor has been described to have a role in the maintenance of G2-M arrest (31). Thus, we considered the possibility that NF-{kappa}B induced p21waf1/cip1 in cell cycle phases other than G1 to regulate G2-M cell cycle arrest, although NF-{kappa}B was activated in each phase of the cell cycle, including the G1 phase, following exposure to IR. To test this notion directly, we sorted G1 cell populations by FACS away from cells in S-G2-M phases based on the DNA content after 6 hours following IR exposure. These cells were then analyzed for the expression of p21waf1/cip1 mRNA by RPA analysis. Surprisingly, the expression of p21waf1/cip1 mRNA was concentrated in the S-G2-M population compared with the G1 cells (Fig. 5B). It was also not induced in CEM-S32/36A cells, confirming NF-{kappa}B dependence. Quantitative real-time PCR showed that the NF-{kappa}B-dependent induction of p21waf1/cip1 mRNA was also seen with another DNA-damaging agent, VP-16 (Fig. 5C). Similar to the results seen with IR, the expression of Bcl-XL was not induced in these cells even with VP-16 treatment (Fig. 5C). Western blotting further confirmed that p21waf1/cip1 protein was indeed induced in a NF-{kappa}B-dependent fashion by both IR and VP-16 as seen by the lack of p21waf1/cip1 protein induction in two different clones expressing the S32/36A superrepressor I{kappa}B{alpha} mutant (Fig. 5D; data not shown). Moreover, NF-{kappa}B-dependent induction of p21waf1/cip1 was not limited to the CEM cell system and was also seen in p53-mutant MDA-MB-231 human breast cancer cells, albeit with much slower kinetics (Figs. 5E and 6). The slower kinetics of p21waf1/cip1 induction in the breast cancer cells may be due to the slower cell cycle kinetics of these cells [doubling time >36 hours at low density (32)] when compared with CEM T leukemic cells that have much shorter doubling time (<24 hours).4 Similar to CEM cells, these breast cancer cells also arrested in G2-M for prolong period on IR exposure, which was reduced when NF-{kappa}B activation was abrogated (Fig. 5F).



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  		FIGURE 5. NF-{kappa}B induces p21waf1/cip1 in CEM and MDA-MB-231 cells. A. CEM{kappa}B and CEM-S32/36A-23 cells were irradiated at a dose of 20 Gy. After 6 hours, total RNA was analyzed by RPA using the hStress-1 template set. Only the sections of the films corresponding to the genes indicated are shown. B. CEM{kappa}B and CEM-S32/36A-23 cells were irradiated at a dose of 20 Gy. After 5.5 hours of incubation, cells were labeled with Hoechst 33342 dye for 30 minutes before sorting the G1 cells from the S-G2-enriched cells on the FACSVantage cell sorter. Total RNA was prepared and RPA was done as above. C. CEM cells were exposed to 10 µmol/L VP-16 for 6 hours followed by quantification of p21waf1/cip1 and Bcl-XL RNA expression levels using real-time reverse transcription-PCR. D. p21waf1/cip1 protein induction was determined by immunoprecipitation-Western blot in both CEM and CEM-S32/36A clones after stimulation by 10 ng/mL TNF-{alpha} (T) or 10 µmol/L VP-16 (V) for 3 hours. E. MDA-MB-231 and MDA-S32/36A-9 cells were treated with 20 Gy IR for up to 72 hours. Total cell extracts were prepared and Western analysis was done using the corresponding antibodies as shown. F. MDA-MB-231 and MDA-S32/36A-9 cells were exposed to 20 Gy IR for up to 72 hours. Cells were prepared as in Fig. 3A and cell cycle profiles were determined at every 24-hour interval.

 


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  		FIGURE 6. MDA-S32/36A clones were unable to induce NF-{kappa}B activation following TNF-{alpha} exposure. MDA-S32/36A clones were tested for their ability to induce NF-{kappa}B following 15-minute TNF-{alpha} (10 ng/mL) stimulation by EMSA (top). Parallel samples were examined for I{kappa}B{alpha} protein levels by Western blot analysis (bottom).

 
NF-{kappa}B Induction of p21waf1/cip1 Contributes to G2-M Arrest
To directly test whether NF-{kappa}B induction of p21waf1/cip1 was critical to regulate G2-M arrest in CEM and MDA-MB-231 cells, we selectively reduced p21waf1/cip1 induction by the use of stable RNA interference. The pSilencer RNA interference vector against p21waf1/cip1 was cotransfected with a puromycin expression vector, and puromycin-resistant clones displaying reduced accumulation of p21waf1/cip1 following IR treatment were isolated (Fig. 7A and C). Both CEM and MDA-MB-231 cells showed a reduction in the number of G2-M-arrested cells and an increase in sub-G1 population indicative of apoptotic cells when p21waf1/cip1 expression was suppressed (Fig. 7B and D). The cell cycle distribution of p21waf1/cip1-silenced MDA-MB-231 cells on IR treatment was nearly identical to that seen with those expressing S32/36A-I{kappa}B{alpha} (compare Figs. 5F and 7D). The reduced inhibitory effect of G2-M arrest in CEM cells was likely due to incomplete silencing efficiency (Fig. 7A). These observations show that NF-{kappa}B-dependent p21waf1/cip1 induction at least partly contributes to G2-M arrest and survival of certain p53-mutant human cancer cell types in response to genotoxic anticancer agents.



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  		FIGURE 7. NF-{kappa}B induction of p21waf1/cip1 contributes to G2-M arrest. A. A pSilencer-p21waf1/cip1 clone (pSilencer-p21-9) and a control pSilencer-scramble clone (pSilencer-Scr) were tested for their ability to reduce p21waf1/cip1 protein expression following 20 Gy IR for 6 hours as above. B. Statistical analysis of three independent experiments showing that pSilencer-scramble cells depict a statistically significant sigmoid curve, indicating that these cells are capable of maintaining a G2-M arrest. In contrast, pSilencer-p21waf1/cip1-9 cells show a statistically significant quadratic polynomial curve, indicating the lack of G2-M maintenance at the 48-hour time point. C. MDA-pSi-p21 cells were tested for their p21waf1/cip protein expression compared with MDA-pSi-Scr control by Western blot analysis following treatment with 20 Gy IR at the indicated times. D. MDA-pSi-Scr and MDA-pSi-p21 cells were treated with 20 Gy IR. Cells were fixed at the indicated time points and cell cycle profiles were determined as in Fig. 3A.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The role of NF-{kappa}B as an apoptosis regulator provided a conceptual framework in which manipulation of NF-{kappa}B activity may serve as a drug target in different pathologic settings, including malignancy (3). Currently, there are several anti-NF-{kappa}B drugs being developed, primarily targeting the proteasome or I{kappa}B kinase activity (33). A common experimental approach to show the role of NF-{kappa}B in cell survival employs the expression of superrepressor I{kappa}B{alpha} or the use of NF-{kappa}B-deficient cells, such as p65 knockout mouse embryonic fibroblasts, to specifically attenuate NF-{kappa}B activity. In this study, we developed a NF-{kappa}B reporter cell system to positively examine the behavior of NF-{kappa}B-activated cells without prior manipulation of NF-{kappa}B activation potential. This system permitted the analysis of populational variations among drug- or radiation-exposed cells with respect to NF-{kappa}B activation, cell cycle regulation, and survival differences.

We found that activation of NF-{kappa}B occurred in different cell cycle phases following stimulation with the DNA-damaging agents, IR and VP-16, with no overt bias toward a specific cell cycle phase. Similar observations were also made with TNF-{alpha} stimulation. In contrast, in accordance with previous findings (20, 21), activation by camptothecin was enriched in the S phase of the cell cycle. These findings provided new conceptual implications regarding the role of NF-{kappa}B in different cell cycle phases. In the case of camptothecin, the data implied that NF-{kappa}B target genes are only induced in S phase or in the later cell cycle phases depending on the kinetics of NF-{kappa}B-dependent target gene expression and duration of NF-{kappa}B activation. In contrast, IR and VP-16 treatments provide the opportunity for NF-{kappa}B to induce its target genes in different cell cycle phases, including the G1 phase, which may be the predominant cell cycle phase in tumor cells in vivo. It was, however, unclear how different NF-{kappa}B target genes were regulated in different cell cycle phases. Our study showed the existence of genes that may be regulated by NF-{kappa}B in a cell cycle phase–selective manner on genotoxic stress insult. We found that p21waf1/cip1 was induced preferentially in cells enriched for S-G2-M populations. In this case, the coupling of p21waf1/cip1 gene expression to specific cell cycle phase (S and G2 but not G1) was apparently critical, because these cells were maintained in G2-M for a longer period to presumably extend the opportunity to repair damaged DNA and eventual cell cycle reentry in the face of DNA damage induction. Thus, these positive selection studies revealed a previously unrecognized G2-M role for NF-{kappa}B to promote cancer cell survival.

The G2-M cell cycle regulation is complex and involves multiple molecular processes under different conditions. These include ataxia telangiectasia mutated, ataxia telangiectasia mutated–related kinase, activation of checkpoint kinases CHK1 and CHK2, phosphorylation of various downstream regulators, such as p53 and CDC25 phosphatases, mismatch repair–dependent processes, etc. (reviewed in refs. 34-37). Induction of p21waf1/cip1 is also implicated in a G2-M arrest in both p53-proficient (38) and p53-deficient (31, 39, 40) cancer cells. Clearly, p21waf1/cip1 has effect on G2-M regulation, because introduction of nonfunctional p21waf1/cip1 or a p21waf1/cip1 antisense oligonucleotide diminished the G2-M arrest phenotype in various cancer cell settings (39, 40). Besides inhibiting cyclin-dependent kinase activity directly to promote cell cycle arrest, interaction of p21waf1/cip1 with proliferating cell nuclear antigen was found to be critical for causing a G2 cell cycle arrest (31). Although p53 induces p21waf1/cip1 expression (23, 38), the mechanism of p21waf1/cip1 induction in p53-deficient cancer cells is not well understood. The results from our current study suggest the possibility that NF-{kappa}B is an important player in p21waf1/cip1 induction in certain p53-defective cancer cell types. A previous study showed that daunomycin-dependent binding of NF-{kappa}B to a putative {kappa}B-binding site in the p21waf1/cip1 promoter correlates with its induction (41). Because p21waf1/cip1 is also implicated as a negative regulator of apoptosis in many cell systems (42), its NF-{kappa}B-dependent induction could also contribute to survival of these G2-M-arrested cells, thereby further contributing to enhanced cancer resistance.

Finally, p21waf1/cip1 is implicated in enhancing NF-{kappa}B-dependent gene expression through the modulation of p300 coactivator activity (43). Thus, induction of p21waf1/cip1 by NF-{kappa}B may create positive feedback stimulation of NF-{kappa}B-dependent transcription in these cancer cells, further promoting their survival. Moreover, NF-{kappa}B activation by DNA-damaging agents seemed to be highly variable with the presence of different levels of cell populations that did not induce NF-{kappa}B-dependent GFP expression. Further dissection of the mechanisms involved in both populational variation of the NF-{kappa}B response and cell cycle–coupled induction of NF-{kappa}B target genes, including p21waf1/cip1, may provide additional insight into understanding cell survival mechanisms provided by this ubiquitous transcription factor.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture and Generation of CEM{kappa}B Cells
CEM human T leukemic and MDA-MB-231 human breast cancer cell lines, along with their derivatives, were maintained in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone Laboratory, Inc., Logan, UT), 100 units penicillin G, and 100 µg/mL streptomycin sulfate (Mediatech). The 3x{kappa}B-GFP reporter was constructed by removing the cytomegalovirus promoter from the pEGFP-C1 plasmid (Clontech, Mountain View, CA) and replacing it with the 3x{kappa}B-tk promoter from the 3x{kappa}B-Luc plasmid (20). The 3x{kappa}B-GFP was introduced into CEM cells by electroporation as described below and selected with 1 mg/mL G418 (Mediatech). Selected CEM cells were then treated with TNF-{alpha} and FACS sorted to isolate the cell clone, CEM{kappa}B, which showed optimal NF-{kappa}B inducibility. Expression of the superrepressor I{kappa}B{alpha} showed the specificity of NF-{kappa}B-dependent GFP induction in the CEM{kappa}B clone (data not shown).

Reagents and Antibodies
A JL Shepherd Model JL-109 with a 137Cs source was used for {gamma} irradiation. Camptothecin, doxorubicin, VP-16, and PI were purchased from Sigma (St. Louis, MO). Human recombinant TNF-{alpha} was purchased from Calbiochem (La Jolla, CA). Actin (C-11), I{kappa}B{alpha} (C-21), p21 (F-5), p21 (C-19), and p53 (D0-1) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase were obtained from Amersham Pharmacia Biotech (Piscataway, NJ), whereas anti-goat antibody conjugated to horseradish peroxidase was obtained from Santa Cruz Biotechnology.

Western Immunoblot Analysis, EMSA, and Immunoprecipitation
Cell preparation and Western blotting were done as described (44). The Ig{kappa}-{kappa}B and Oct-1 probes and conditions for EMSA were as described (45). To detect p21waf1/cip1 protein in CEM{kappa}B cells, 5 x 106 cells were lysed in 10% PBS and 90% lysis buffer as described previously (46). Supernatants were diluted further in lysis buffer, and p21 (C19) antibody (1 µg) was added to each tube. Samples were rotated for 60 minutes at 4°C. Protein G-Sepharose beads (Amersham Pharmacia Biotech) were then added to each tube, and the samples were rotated overnight at 4°C. p21waf1/cip1 protein was resolved in 12% SDS-PAGE gels and analyzed by Western blotting using the same antibody.

FACS Sorting, Cell Cycle Analysis, and Fluorescent Microscopy
CEM{kappa}B cells were exposed to 10 Gy IR and allowed to repair for a total of 6 hours at 37°C. GFP-positive CEM{kappa}B cells were sorted from GFP-negative cells using the FACSVantage cell sorter (BD PharMingen, San Jose, CA) followed by immediate fixation in 70% ethanol. For cell cycle analysis, cells were processed as described previously (47) and analyzed on a FACScan flow cytometer (BD PharMingen). Data were analyzed using the CellQuest (BD PharMingen) and ModFit (Verity Software House, Topsham, ME) software. CEM{kappa}B cells were visualized and photographed as described previously (48).

RNase Protection Assay
CEM{kappa}B and CEM-S32/36A cells were irradiated at a dose of 20 Gy. Following a 5.5-hour incubation, cells were labeled for 30 minutes with Hoechst 33342 dye (Molecular Probes, Eugene, OR) before sorting on the FACSVantage cell sorter. Cells in the G1 phase of the cell cycle were sorted from the total of the population. Cells were collected at 4°C, washed in PBS, and immediately frozen at –70°C. Cells were homogenized using a Qiashredder column (Qiagen, Valencia, CA) followed by RNA isolation using the RNeasy kit (Qiagen). The RiboQuant RPA kit (BD PharMingen) was used to perform RPA on the isolated RNA by hybridizing it with the hStress-1 multiprobe template set (BD PharMingen) as outlined in the manufacturer's directions.

Quantitative Reverse Transcription-PCR Analysis
Total RNA from CEM cells treated with 10 µmol/L VP-16 for 6 hours was extracted using the RNeasy kit and Qiashredder. cDNA was synthesized as described (49). Quantitative real-time reverse transcription-PCR reactions (25 µL) contained 2 µL cDNA, 12.5 µL SYBR Green (Applied Biosystems, Foster City, CA), and the appropriate primers. Product accumulation was monitored by SYBR Green fluorescence. The relative expression levels were determined from a standard curve of serial dilutions of cDNA samples. Forward and reverse primers for real-time PCR were (1) human p21waf1/cip1 5'-GCAGACCAGCATGACAGATTTC-3' and 5'-GCGGATTAGGGCTTCCTCTT-3', human Bcl-XL 5'-TGCCTAAGGCGGATTTGAAT-3' and 5'-ATTGTCCAAAACACCTGCTCACT-3', and glyceraldehyde-3-phosphate dehydrogenase primer: 5'-GAAGGTCGGAGTCAACGGATTT-3' and 5'-GAATTTGCCATGGGTGGAAT-3'.

Generation of Stable p21waf1/cip1 pSilencer Knockdown Clones
p21waf1/cip1 RNA interference stable CEM and MDA clones were generated using the pSilencer vector (Ambion, Austin, TX). A 19-nucleotide RNA interference sequence was chosen to knockdown p21waf1/cip1 that had no significant homology to any other gene in the human genome. Two DNA oligonucleotides were designed for knocking down p21waf1/cip1 following the manufacturer's protocol linking the 19-nucleotide sense and antisense sequences as follows: 5'-CTTCGACTTTGTCACCGAGTTCAAGAGACTCGGTGACAAAGTCGAAGTTTTTT-3'(sense) and 5'-AATTAAAAAACTTCGACTTTGTCACCGAGTCTCTTGAACTCGGTGACAAAGTCGAAGGGCC-3' (antisense). The scramble control was made as follows: 5'-TACCGTCTCCACTTGATCGTTCAAGAGACGATCAAGTGGAGACGGTATTTTTT-3'(sense) and 5'-AATTAAAAAATACCGTCTCCACTTGATCGTCTCTTGAACGATCAAGTGGAGACGGTAGGCC-3' (antisense). The oligonucleotides were annealed, and the resulting insert was ligated into the pSilencer vector that had been linearized with the restriction enzymes ApaI and EcoRI. The resulting plasmid (40 µg) and a puromycin resistance vector pLPL-CA (4 µg) were cotransfected into both CEM and MDA cells by electroporation at a setting of 300 V and 950 µF in a Bio-Rad (Hercules, CA) Gene Pulser apparatus with capacitance extender. Stable clones were selected by 1 µg/mL puromycin and screened for their ability to knockdown p21waf1/cip1 protein expression by Western blotting.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Kathy Schell for help with FACS analyses, Bradley Seufzer for technical support, and Randy Tibbetts and the Miyamoto laboratory members for helpful discussion and critical reading of the article.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Department of Defense grant BC010767 (S.M. Wuerzberger-Davis), American Heart Association predoctoral fellowship 0310015Z (P-Y. Chang), NIH grants R01-CA77474 and R01-CA81065, and Shaw Scientist Award from the Milwaukee Foundation (S. Miyamoto).

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.

4 Wuerzberger-Davis, unpublished observations. Back

Received 3/10/05; revised 4/29/05; accepted 5/ 2/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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