Molecular Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Basile, J. R.
Right arrow Articles by Münger, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Basile, J. R.
Right arrow Articles by Münger, K.
Molecular Cancer Research 1:262-270 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

NF-{kappa}B-Mediated Induction of p21Cip1/Waf1 by Tumor Necrosis Factor {alpha} Induces Growth Arrest and Cytoprotection in Normal Human Keratinocytes1

John R. Basile1,2,2, Alexandra Eichten1, Valerie Zacny1 and Karl Münger1

1 Department of Pathology, Harvard Medical School, Boston, MA and
2 Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, MA

Requests for reprints: Karl Münger, Department of Pathology, Harvard Medical School, 200 Longwood Avenue, D2/544A, Boston, MA 02115-5701. Phone: (617) 432-2878; Fax: (617) 432-0426. E-mail: karl_munger{at}hms.harvard.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Cellular stressors such as UV irradiation, chemical irritants, or an immune system challenge in an otherwise healthy host induce the production and release of cytokines, such as tumor necrosis factor (TNF) {alpha}, which are powerful regulators of tissue homeostasis. TNF{alpha}, an important mediator of inflammation in the skin and mucosa, often represents the first physiological response to such noxious stimuli. TNF{alpha} not only acts systemically to promote inflammation, but also locally at the site of the stimulus to modulate cell growth and survival. It has been demonstrated previously that epithelial cells undergo growth arrest and differentiation in the presence of TNF{alpha}. However, the mechanism of this response is not well understood. Here we show that in primary cultures of human foreskin keratinocytes, TNF{alpha} mediates cellular growth arrest through activation of the transcription factor NF-{kappa}B. The cdk inhibitor p21Cip1/Waf1 is activated through NF-{kappa}B and is an important mediator of this growth arrest response. In addition, TNF{alpha}-treated cell populations are markedly less susceptible to apoptosis by UV irradiation and this cytoprotective effect is at least in part mediated by p21Cip1/Waf1 as well.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Skin and mucosa are continually subjected to harmful agents, such as microorganisms, physical and chemical injury, and UV irradiation, which stimulate the production and release of cytokines (1). Cytokines are powerful mediators of immunity produced by macrophages, fibroblasts, and even epithelial cells themselves (2). Tumor necrosis factor (TNF) {alpha}, a member of a group of related cytokines called the TNF{alpha} superfamily, is one of the main mediators of inflammation in the skin and mucosa (3).

Originally identified as a protein that caused hemorrhagic necrosis of transplanted tumor tissue (4), TNF{alpha} was subsequently discovered to be identical to the catabolic protein cachectin, a substance associated with fever and wasting in cancer patients (5). TNF{alpha} plays an important role in the acute phase reaction, modulating fever and inflammation by activating the strongly pro-inflammatory transcription factor NF-{kappa}B (reviewed in Ref. 6). In addition to its numerous systemic effects, TNF{alpha} and related family members affect individual epithelial cells by binding to cell surface receptors and modulating proliferation, differentiation, or apoptosis (3).

TNF{alpha} exerts its effects in target cells by binding to its cell surface receptors, TNF{alpha} receptors 1 and 2 (TNF-R1 and -R2) (7). When bound by TNF{alpha}, TNF-R1 forms a trimer and recruits an adaptor protein, TNF-R1-associated death domain protein (TRADD), to its carboxyl-terminal cytoplasmic tail, termed the death domain (8). While devoid of intrinsic enzymatic activity, TRADD couples the receptor to two separate and often opposing signaling pathways by binding to different adapter proteins. Interaction with the Fas-associating protein and death domain (FADD) mediates activation of the caspase cascade, whereas binding to the TNF-R1-associated factor 2 (TRAF2) and receptor interacting protein (RIP) results in NF-{kappa}B activation (9, 10).

FADD is a Mr 23,000 protein that upon recruitment by TRADD binds pro-caspase 8 via its amino-terminal death effector domain. This results in autocatalytic activation of pro-caspase 8 (11), which in turn cleaves substrates including downstream pro-caspases, eventually resulting in the activation of effector caspases and apoptosis (12). Simultaneously, the binding of TRADD to the adapters TRAF2 and RIP induces phosphorylation and activation of the NF-{kappa}B essential modifier (NEMO). NEMO (IKK{gamma}) is a regulatory protein that links upstream activators such as TNF{alpha} to IKK{alpha} and IKKß, the two subunits of the kinase involved in the phosphorylation of the NF-{kappa}B inhibitor, I-{kappa}B (13–15). I-{kappa}B, which normally binds and inactivates NF-{kappa}B by keeping it sequestered in the cytoplasm (13, 16–18), undergoes proteosome-mediated degradation when phosphorylated. This event results in nuclear translocation of NF-{kappa}B and initiates the transcription of numerous pro-inflammatory genes, a response strongly associated with cell survival (reviewed in Ref. 6).

TNF-R2, the other major cell surface receptor for TNF{alpha}, does not contain a death domain and therefore does not directly signal apoptosis (19). Instead, TNF{alpha} binding to TNF-R2 recruits TRAFs to the receptor (20), sometimes resulting in the titration of these protective adapters away from TNF-R1 and actually enhancing cell death (reviewed in Ref. 21). It is important to note, however, that TNF-R2 is relatively insensitive to soluble TNF{alpha} in vitro due to the weak interaction between the receptor and TRAF2 and instead responds better to membrane-bound TNF{alpha} (22).

Clearly, the cellular consequences of TNF-R1 stimulation are critically dependent upon the cell type and experimental conditions used. This is illustrated by the varied cellular responses to TNF{alpha} that have been observed. These range from induction of apoptosis to NF-{kappa}B-mediated activation of pathways that alter cell growth and differentiation. Our group and others have demonstrated that TNF{alpha} treatment of primary cultures of human keratinocytes results in growth arrest rather than apoptosis (23–28). This is consistent with other studies demonstrating that without concurrent suppression of new protein synthesis, many cell types are resistant to TNF{alpha}-mediated apoptosis (29).

Despite the involvement of NF-{kappa}B in regulating the cytostatic effect of TNF{alpha}, the mechanism of this response is not well understood. Here we show that the ability of TNF{alpha} to induce cytostasis in primary human epithelial cells is due to NF-{kappa}B-mediated induction of the cdk inhibitor p21Cip1/Waf1. Moreover, TNF{alpha} triggers a pronounced anti-apoptotic response, and NF-{kappa}B-mediated induction of p21Cip1/Waf1 also represents an important aspect of the anti-apoptotic activity of TNF{alpha} in primary human foreskin keratinocytes (HFKs).


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
NF-{kappa}B Activation Is Necessary for the Growth Arrest Response of Primary Human Epithelial Cells to TNF{alpha}
To test whether the growth arrest response of TNF{alpha} in primary human epithelial cells is mediated by activation of NF-{kappa}B, we inhibited NF-{kappa}B activity in HFKs by treating cells with the NEMO binding domain peptide (NBD) and ectopic expression of the unphosphorylatable I-{kappa}B "super-repressor" mutant {Delta}N-I-{kappa}B.

NBD is a cell-permeable peptide that mimics the NEMO binding domain of IKKß, thereby disrupting its interaction with endogenous NEMO and preventing I-{kappa}B phosphorylation and hence activation of NF-{kappa}B (30). To determine the specificity of NF-{kappa}B inhibition by NBD, we analyzed the generation of phosphorylated I-{kappa}B, and the phosphorylated, active forms of JNK/SAPK and p38 that are also activated in HFKs by TNF{alpha} treatment (23). Control HFK cultures incubated with TNF{alpha} and scrambled NBD peptide showed transient appearance of phospho-I-{kappa}B, a pattern reflecting its phosphorylation and degradation along with the normal phosphorylation of p38 and JNK (Fig. 1A). In contrast, there was no evidence for phospho-I-{kappa}B formation and degradation in cells co-treated with NBD peptide and TNF{alpha} (Fig. 1A). Treatment with the NBD peptide, however, did not affect the kinetics of induction of p38 and JNK/SAPK phosphorylation (Fig. 1A). As expected, inhibition of NF-{kappa}B activation by this peptide resulted in prolonged maintenance of the phosphorylated, active forms of p38 and JNK (Fig. 1A). Taken together, these results indicate that the NBD peptide specifically inhibits TNF{alpha}-mediated NF-{kappa}B activation.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1. A. NBD peptide specifically inhibits NF-{kappa}B activation by TNF{alpha}. HFKs were incubated with 10 ng/ml TNF{alpha} with or without DMSO, 200 µM wild-type NBD peptide, or the scrambled control peptide for the indicated periods of time and analyzed for the appearance of phosphorylated I-{kappa}B, p38, and JNK/SAPK. GAPDH was used as a loading control (lower panel). B. NBD peptide inhibits TNF{alpha}-mediated growth inhibition. HFKs were grown in the presence of DMSO, 50 µM wild-type NBD peptide (NBD), or the scrambled NBD peptide (NBDscr) with or without 10 ng/ml TNF{alpha} for 24 h. Cell cycle fractions were determined by FACS analysis of propidium iodide-stained cells. G0-G1, S, and G2-M fractions are expressed as the percentages of cells pooled from three independent experiments. C. {Delta}N-I-{kappa}B inhibits TNF{alpha}-mediated growth inhibition. HFKs transiently expressing either pMIG vector or pMIG/{Delta}N-I-{kappa}B were grown in the presence or absence of 50 ng/ml TNF{alpha} for 24 h. Cell cycle distribution of transfected cells was determined by two-dimensional FACS analysis of propidium iodide-stained cells that were also positive for GFP expression. Results are presented as percentage increase of transfected cells in G0-G1.

 
To determine if NF-{kappa}B inhibition may affect the TNF{alpha}-mediated cytostatic response, subconfluent HFK cultures were incubated with either wild-type NBD peptide, the scrambled NBD control peptide, or solvent (DMSO) alone, with or without 10 ng/ml TNF{alpha}. After 24 h, cell cycle distribution was determined by fluorescence-activated cell sorting (FACS) analysis. As expected, TNF{alpha} treatment caused a G1 growth arrest as evidenced by higher G0-G1 and lower S-phase fractions, compared to non-cytokine-treated cells (Fig. 1B, upper right panel). Inhibition of NF-{kappa}B activation by NBD peptide, however, abrogated this TNF{alpha}-induced growth arrest (Fig. 1B, lower right panel). This effect was specific because cells treated with scrambled NBD control peptide remained susceptible to TNF{alpha}-mediated growth inhibition (Fig. 1B, middle right panel).

To confirm these results by an independent technique, bi-cistronic vectors coding for GFP alone (pMIG) or the I-{kappa}B super-repressor plus GFP (pMIG/{Delta}N-I-{kappa}B) were transfected into HFKs. The cells were grown in either normal keratinocyte serum-free media or media containing 50 ng/ml of TNF{alpha}. The cell cycle distribution of transfected, GFP-positive cells was determined by FACS. As expected, TNF{alpha} treatment of control HFKs transfected with the empty pMIG vector caused an increase in the G0-G1 population (Fig. 1C). In contrast, the pMIG/{Delta}N-I-{kappa}B-expressing populations were resistant to TNF{alpha}-mediated growth arrest and no accumulation of G0-G1 cells was observed (Fig. 1C).

Taken together, these results indicate that NF-{kappa}B activation is crucial in mediating the TNF{alpha}-induced G1 growth arrest in primary human epithelial cells.

NF-{kappa}B Mediated Induction of p21Cip1/Waf1 by TNF{alpha}
We have previously demonstrated that TNF{alpha}-mediated growth arrest is accompanied by a modest induction of the cdk inhibitor p21Cip1/Waf1 (23). To determine whether the increased p21Cip1/Waf1 expression upon TNF{alpha} treatment is NF-{kappa}B dependent, HFKs were co-transfected with a p21Cip1/Waf1 promoter-driven luciferase reporter or the empty control plasmid and the cells treated with TNF{alpha}. A strong activation of the reporter was observed both at 12 and 24 h after TNF{alpha} treatment, indicating that the p21Cip1/Waf1 promoter is activated by this cytokine (Fig. 2A). To determine whether this induction is dependent upon NF-{kappa}B activation, we performed co-transfection experiments with vectors encoding the {Delta}N-I-{kappa}B super-repressor or a dominant negative TRADD mutant (DN-TRADD) incapable of interacting with TRAF2 and activating NF-{kappa}B. TNF{alpha}-mediated activation of the reporter was effectively abolished at 12 h and strongly inhibited at 24 h by co-expression of {Delta}N-I-{kappa}B and DN-TRADD. These results strongly suggest that activation of the p21Cip1/Waf1 reporter construct by TNF{alpha} in HFKs is through NF-{kappa}B activation (Fig. 2A).



View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 2. A. TNF{alpha} activates p21Cip1/Waf1 transcription in HFKs in a TRADD- and NF-{kappa}B-dependent manner. HFKs were co-transfected with plasmids containing a p21Cip1/Waf1 promoter-driven luciferase (pWpsLuc) or the parental vector (pBL), along with either empty vector or vector coding for DN-TRADD, DN-TRAF2, or {Delta}N-I-{kappa}B, followed by treatment with 10 ng/ml TNF{alpha} for 12 and 24 h. Each transfection also contained TK-promoter-driven renilla luciferase as a transfection control. B. TNF{alpha}-mediated increases in p21Cip1/Waf1 levels are dependent on NF-{kappa}B activation. HFKs were treated with 10 ng/ml TNF{alpha} with or without DMSO, 200 µM wild-type NBD peptide, or the scrambled control peptide for the indicated periods of time. Steady-state levels of p21Cip1/Waf1 were determined by immunoblot analysis. Actin is used as a loading control (lower panel). Relative levels of p21Cip1/Waf1 normalized for actin expression are presented by the bar graph.

 
To determine whether NF-{kappa}B mediates the observed increases in p21Cip1/Waf1 levels upon TNF{alpha} treatment, HFKs were incubated with TNF{alpha} in combination with the NBD peptide. Control cells were treated in parallel with TNF{alpha} and solvent (DMSO) or scrambled NBD peptide, and the levels of p21Cip1/Waf1 were evaluated by immunoblot analysis. As reported previously (23) TNF{alpha} caused a 2-fold increase of p21Cip1/Waf1 levels over a time period of 96–120 h, and this response was largely abrogated in HFKs incubated with TNF{alpha} and NBD peptide (Fig. 2B).

These results support the model that TNF{alpha}-mediated induction of p21Cip1/Waf1 in HFKs is critically dependent on NF-{kappa}B activation.

Induction of p21Cip1/Waf1 Is Necessary for TNF{alpha}-Mediated Growth Arrest in HFKs
After demonstrating that NF-{kappa}B activity is necessary for TNF{alpha}-induced growth arrest and that p21Cip1/Waf1 induction is through an NF-{kappa}B-dependent pathway, we next determined whether p21Cip1/Waf1 is necessary to induce TNF{alpha}-mediated growth arrest in keratinocytes. HFKs were transfected with a p21Cip1/Waf1-specific double-stranded small inhibitory RNA (siRNA) oligonucleotide to inhibit p21Cip1/Waf1 expression or a scrambled control oligonucleotide and treated with TNF{alpha} for 48 h. As expected, immunofluorescence analysis revealed fewer nuclei staining positive for p21Cip1/Waf1 in the wild-type siRNA-transfected cells than in cells transfected with the scrambled control oligonucleotide (Fig. 3A). Flow cytometric analysis of the different HFK populations showed that the growth arrest response to TNF{alpha} was decreased in wild-type siRNA-transfected cells compared to cells transfected with control oligonucleotide (Fig. 3B). These results demonstrate that p21Cip1/Waf1 is an important mediator of TNF{alpha} cytostasis in HFKs.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 3. A. RNA interference inhibits TNF{alpha}-mediated increases in p21Cip1/Waf1 in HFKs. HFKs transfected with double-stranded small inhibitory (si)RNA complementary to a portion of the p21Cip1/Waf1 gene (wt) or a scrambled control oligonucleotide (scr) were treated with 10 ng/ml TNF{alpha} for 48 h and processed for immunofluorescence using a rhodamine red-coupled secondary antibody. Nuclei were counterstained with bisbenzimide (Hoechst). The pictures shown in the panels have been electronically inverted to increase contrast. B. Increases in p21Cip1/Waf1 levels are responsible for TNF{alpha}-mediated growth inhibition. HFKs transfected with wild-type or scrambled double-stranded p21Cip1/Waf1 siRNA were treated with 10 ng/ml TNF{alpha} for 48 h and processed for propidium iodide FACS analysis of DNA content (upper panel). Percentages of cells in G0-G1, S, and G2-M phases of the cell cycle are shown graphically (lower panel).

 
Inhibition of NF-{kappa}B Activation Causes TNF{alpha}-Mediated Apoptosis
In many cell types, including HFKs, the pro-apoptotic activity of TNF{alpha} requires inhibition of new protein synthesis by cycloheximide (29, 31, 32). This presumably allows for the intracellular clearance of short-lived anti-apoptotic molecules such as the cFLIPS (33, 34) and/or to restrain production of pro-survival factors via NF-{kappa}B (reviewed in Ref. 6). To examine if inhibition of NF-{kappa}B activation can unmask the apoptotic response of HFKs to TNF{alpha}, cells were incubated with wild-type NBD peptide and TNF{alpha} for 24 or 48 h with or without the protein synthesis inhibitor CHX. Control cells were treated in parallel with TNF{alpha} and scrambled NBD peptide or solvent (DMSO). Incubation of HFKs with TNF{alpha} in combination with NBD peptide triggered an apoptotic response in the absence of protein synthesis inhibition by CHX (Fig. 4A). Consistent with reports that NF-{kappa}B also exhibits anti-apoptotic properties that appear to be independent of new protein synthesis (35), an even more dramatic effect of NF-{kappa}B inhibition was observed when TNF{alpha} was administered in combination with CHX (Fig. 4A).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. A. NF-{kappa}B activation by TNF{alpha} protects against its own apoptotic signaling. HFKs were grown in DMSO, 200 µM wild-type NBD (NBDwt), or the scrambled NBD peptide (NBDscr) with or without 10 ng/ml TNF{alpha} and 30 mg/ml CHX for 24 and 48 h. Apoptosis was determined by assessment of nuclear morphology using Hoechst staining. The bar graphs are the results from three independent experiments. B. p21Cip1/Waf1 induction does not markedly alter TNF{alpha} apoptotic signaling. HFKs transfected with wild-type (wt) or scrambled (scr) double-stranded p21Cip1/Waf1 siRNA and treated with or without TNF{alpha} and 30 mg/ml CHX for 24 h. Apoptosis was determined by nuclear morphology using Hoechst staining. The bar graphs are the results from three independent experiments.

 
NF-{kappa}B-induced expression of p21Cip1/Waf1 plays an important role in the growth inhibitory response to TNF{alpha} (Fig. 3). Because p21Cip1/Waf1 and the CKIs in general have been associated not only with growth arrest and differentiation but also with resistance to cell death (36–39), we next examined whether p21Cip1/Waf1 induction is also linked to the cytoprotective, anti-apoptotic activity of TNF{alpha} in HFKs. Cells were incubated with TNF{alpha} for 24 h with or without CHX and either wild-type or scrambled control p21Cip1/Waf1 siRNA oligonucleotides. In contrast to inhibition of NF-{kappa}B activation (Fig. 4A), inactivation p21Cip1/Waf1 did not trigger an apoptotic response to TNF{alpha} in the absence of protein synthesis inhibition (Fig. 4B). Apoptosis was only slightly enhanced upon p21Cip1/Waf1 inhibition in HFKs treated with TNF{alpha} and CHX (Fig. 4B). These results suggest that TNF{alpha}-mediated induction of p21Cip1/Waf1 and the ensuing growth arrest is to a large part separable, and not sufficient for NF-{kappa}B-mediated cytoprotection to TNF{alpha} in HFKs.

TNF{alpha} Causes NF-{kappa}B-Mediated Apoptosis Resistance to UV Irradiation
Keratinocytes exposed to UV light suffer DNA damage and undergo either growth arrest or cell death (40). To test whether TNF{alpha} can elicit an anti-apoptotic response in cells exposed to pro-apoptotic stimuli such as UV, and to establish the role of NF-{kappa}B in this process, HFKs were grown for 24 h in media containing TNF{alpha} and wild-type NBD peptide. Control cells were treated in parallel with TNF{alpha} and scrambled NBD peptide or solvent (DMSO). Cells were then UV irradiated, returned to normal media, and evaluated for apoptosis the following day. UV irradiation of non-TNF{alpha}-pretreated HFKs resulted in 24.5% apoptosis, and this response was not greatly altered in cells incubated with either the wild-type or scrambled NBD peptide (21.8% and 25.8% apoptosis, respectively, Fig. 5A). Cells pretreated with TNF{alpha} for 24 h, however, were markedly less susceptible to UV-induced apoptosis (13.3%). This anti-apoptotic, cytoprotective activity of TNF{alpha} was abrogated when NF-{kappa}B activation was inhibited by NBD peptide (30% apoptosis). In contrast, the scrambled mutant NBD peptide afforded no protection (15.1% apoptosis) (Fig. 5A). These results indicate that TNF{alpha} elicits a cytoprotective response in HFKs that is dependent upon NF-{kappa}B activation.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. A. TNF{alpha}-mediated activation of NF-{kappa}B protects against UV-mediated apoptosis. Normal HFKs were pretreated for 24 h with the indicated media and irradiated for 3 min in a tissue culture hood with the bactericidal UV lamp. Normal medium was added back to the plates for 24 h after which the cells were fixed in methanol and stained with Hoechst 33258. Apoptotic nuclei were counted as a percentage of total nuclei with the data points representing the averages of three experiments. B. p21Cip1/Waf1 inhibition partly abrogates TNF{alpha}-mediated protection against UV-induced apoptosis. Normal HFKs transfected with wild-type (wt) or scrambled (scr) double-stranded p21Cip1/Waf1 siRNA were irradiated at 24 h post transfection for 3 min in a tissue culture hood with the bactericidal UV lamp. Normal medium was added back to the plates for 24 h, after which the cells were fixed in methanol and stained with Hoechst 33258. Apoptotic nuclei were counted as a percentage of total nuclei with the data points representing the averages of three experiments.

 
Next we determined whether NF-{kappa}B-mediated induction of p21Cip1/Waf1 might contribute to the cytoprotective effect of TNF{alpha} to UV-induced apoptosis. HFKs transfected with either p21Cip1/Waf1-specific siRNA oligonucleotide or the scrambled control oligonucleotide were grown for 24 h in TNF{alpha}-containing media, exposed to UV irradiation, and evaluated for apoptosis 24 h thereafter. Inhibition of p21Cip1/Waf1 expression strongly compromised the NF-{kappa}B-mediated cytoprotective effect of TNF{alpha} to apoptosis by UV irradiation (Fig. 5B). These results indicate that p21Cip1/Waf1 plays an important role in mediating the anti-apoptotic effect of TNF{alpha} to UV irradiation.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Ligation of TNF{alpha} to its cognate receptor TNF-R1 results in the recruitment of the adaptor protein TRADD. TRADD not only triggers the formation of the death-inducing signaling complex (DISC) which initiates the caspase cascade, but also associates with TRAF2 which activates mostly anti-apoptotic pathways through NF-{kappa}B and the MAP kinase family members p38 and JNK. Consequently, activation of TNF-R1 results in apoptosis only under certain conditions and in specific cell types (reviewed in Ref. 12). We have previously demonstrated that TNF{alpha}-treated HFKs undergo p53-independent G1 growth arrest accompanied by increased expression of the cdk inhibitor p21Cip1/Waf1 and differentiation markers such as transglutaminase (23). It is conceivable that in a squamous epithelium where cells are constantly and rapidly turned over through the natural process of differentiation, denucleation, and sloughing off of terminally differentiated squames, induction of premature growth arrest and differentiation may represent a functional alternative to induction of apoptosis of abnormal cells in other cell types.

The results presented here demonstrate that the TNF{alpha}-induced G1 growth arrest response in HFKs is largely mediated through NF-{kappa}B (Fig. 1). These findings are consistent with the model that NF-{kappa}B plays a unique role in epithelial cell differentiation and proper development of the epidermis and that differentiation of normal human keratinocytes may be controlled by NF-{kappa}B target genes. Indeed, NF-{kappa}B inhibition results in epithelial hyperplasia in transgenic mice (41–44), and ectopic expression of c-Rel can induce cell cycle arrest in the cervical adenocarcinoma cell line HeLa (45). NF-{kappa}B regulates the transition of the rapidly proliferating undifferentiated basal cells to the less proliferative, differentiated cells of the suprabasal compartment (41, 46), and the human papillomavirus E7 oncoprotein, which can uncouple cellular proliferation and differentiation in human keratinocytes (47), affects NF-{kappa}B activation by targeting the I-{kappa}B kinase complex (48). Even though TNF{alpha} treatment of IMR90 normal human diploid fibroblasts induces rapid and efficient NF-{kappa}B activation, no growth arrest is observed (23, 49). These results indicate that other epithelial specific transcription factors cooperate with NF-{kappa}B to induce a growth arrest/differentiation response in HFKs.

Consistent with previously published results that p21Cip1/Waf1 expression may be regulated by NF-{kappa}B (46, 50, 51), we found that TNF{alpha}-mediated induction of expression of the cdk inhibitor p21Cip1/Waf1 in HFKs is through NF-{kappa}B activation (Fig. 2). Even though several putative NF-{kappa}B binding sites have been identified in the p21Cip1/Waf1 promoter (52), the exact mechanisms of transactivation of the p21Cip1/Waf1 promoter by NF-{kappa}B are not yet well understood. Hence, it is possible that NF-{kappa}B induces p21Cip1/Waf1 levels through an indirect mechanism. Consistent with such an indirect mechanism, it has been reported that transcription of the NF-{kappa}B-family member RelB is regulated by NF-{kappa}B and that RelB can increase the expression of p21Cip1/Waf1 protein at the transcriptional level (53).

Our result that inhibition of p21Cip1/Waf1 expression by siRNA oligonucleotides inhibits growth suppression by TNF{alpha} (Fig. 3), however, is the first demonstration that p21Cip1/Waf1 plays an important role in mediating the cytostatic activity of TNF{alpha} in primary human epithelial cells. This is all the more surprising because the induction of p21Cip1/Waf1 in HFKs in response to TNF{alpha} treatment is only approximately 2-fold (Ref. 23 and Fig. 2B), and thus considerably less dramatic than in response to a p53-induced growth arrest stimulus. The cdk inhibitor p21Cip1/Waf1 is intimately involved in coupling growth arrest to cellular differentiation in epithelial cells (54–56) and the level of induction of p21Cip1/Waf1 expression during calcium-induced differentiation of HFKs (55) is similar to that observed here (Fig. 2B) and in a previous study (23) in response to TNF{alpha}. It remains to be determined, however, whether under normal physiological conditions of keratinocyte differentiation, p21Cip1/Waf1 induction is also mediated through NF-{kappa}B. The absence of an overt skin differentiation phenotype in p21Cip1/Waf1-deficient mice (57) suggests that unlike in TNF{alpha}-treated human keratinocytes, other molecules may compensate for the loss of p21Cip1/Waf1 expression in normal differentiation of murine skin.

Inhibition of NF-{kappa}B activation by the cell-permeable NBD peptide that mimics the NEMO binding domain of IKKß and hence interferes with NF-{kappa}B activation (30) renders HFKs susceptible to apoptosis by TNF{alpha} even in the absence of protein synthesis inhibitors (Fig. 4A). This result clearly illustrates the importance of NF-{kappa}B in mounting an anti-apoptotic response, which is dominant in normal human keratinocytes. This effect is not solely due to transcription of NF-{kappa}B target genes, because NF-{kappa}B inhibition also enhanced apoptosis when TNF{alpha} was administered to cells in combination with the protein synthesis inhibitor CHX (Fig. 4A). These findings are consistent with the finding that some of the anti-apoptotic effects of NF-{kappa}B may be independent of new protein synthesis (35). The transcription-independent anti-apoptotic activity of NF-{kappa}B may also in part explain our finding that treatment of HFKs with TNF{alpha} and CHX yields only a modest apoptotic response compared to TRAIL or agonistic Fas antibody administered in combination with CHX which do not trigger NF-{kappa}B activation in this cell type (23).

Even though inhibition of NF-{kappa}B is sufficient to unmask the apoptotic response of HKFs to TNF{alpha} in the absence of protein synthesis inhibition by CHX (Fig. 4A), inhibition of p21Cip1/Waf1 induction by siRNA oligonucleotides does not result in a similar apoptotic response (Fig. 4B). These results suggest that whereas p21Cip1/Waf1 is a critical NF-{kappa}B target that mediates the cytostatic effect of TNF{alpha} in HFKs (Fig. 3B), it is not a rate-limiting component of TNF{alpha}-induced, NF-{kappa}B-mediated pro-survival signaling that balances the pro-apoptotic DISC response upon TNF-R1 activation (Fig. 4B).

In contrast, however, TNF{alpha} was able to protect cells from UV-induced apoptosis. This cytoprotective effect of TNF{alpha} was also dependent on NF-{kappa}B signaling (Fig. 5A). Interestingly, p21Cip1/Waf1 inhibition to a large extent abrogated the NF-{kappa}B-mediated protection that TNF{alpha} afforded to UV-induced apoptosis in HFKs (Fig. 5B). Immunofluorescence analysis revealed a predominantly nuclear staining pattern of p21Cip1/Waf1 in TNF{alpha}-treated HFKs, suggesting that it might not function as a direct inhibitor of caspase 3 as has been suggested by other studies (58), and thus, its anti-apoptotic action may be directly related to its growth suppressive activity.

In summary, our results demonstrate that NF-{kappa}B plays a central role in inducing a p53-independent growth arrest and protection from apoptosis in primary human epithelial cells. The growth arrest response is largely mediated by induction of the cdk inhibitor p21Cip1/Waf1, but induction of this protein plays only a minor role in suppressing TNF{alpha}-induced apoptosis. Thus, TNF{alpha}-mediated NF-{kappa}B activation induces several independent cytoprotective pathways. Some may specifically protect against TNF{alpha}-mediated (and other death domain receptor-mediated) apoptosis, presumably at the level of DISC-mediated caspase 8 activation. Examples of such molecules include cFLIP (59, 60), c-IAPs (61), or the NF-{kappa}B-inducible DED-containing protein NDED (62). Induction of p21Cip1/Waf1 in TNF{alpha}-treated keratinocytes, however, inhibits their susceptibility to other apoptotic stimuli such as UV irradiation (Fig. 5B).


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture
Primary HFKs were prepared from a pool of several neonatal foreskins obtained after routine circumcision, following the protocol of Rheinwald and Beckett (63). Cells were maintained in serum-free keratinocyte growth medium (Life Technologies, Inc., Carlsbad, CA, Keratinocyte-SFM, 10724-011) supplemented with 100 units/ml penicillin/streptomycin (Life Technologies, Inc., 15070-063), 0.5 µg/ml Fungizone (Gemini Bio-Products, Calabasas, CA, 400-104), and 0.1 mg/ml gentamycin (Life Technologies, Inc., 150710-064).

UV Treatment
A subconfluent 35-mm plate of HFKs with the medium removed was placed 6 in. from a bactericidal UV light and irradiated for 3 min. Normal medium was returned to the plate and the cells were allowed to grow overnight at 37°C and 7% CO2.

Keratinocyte Transfection
HFKs were transfected using Fugene (Roche, Indianapolis, IN) according to the manufacturer's instructions. Briefly, for each transfection, 90 µl of KSFM was mixed with 10 µl of Fugene and incubated at room temperature for 5 min. Eight micrograms of total DNA was added to this mixture, incubated for 15 min at room temperature, and this solution was evenly added to a 35-mm plate of subconfluent HFKs in 1 ml KSFM. The cells were left in transfection mixture overnight at 37°C and 7% CO2, then washed in PBS and returned to fresh KSFM the following day.

Cytokine and Peptide Treatment
Subconfluent HFK cultures were treated with the indicated concentrations of TNF{alpha} (R&D Systems, Minneapolis, MN) and were noted with 30 µg/ml of cycloheximide (Sigma, St. Louis, MO) for the indicated periods of time. Fresh TNF{alpha}-containing medium was added to the cultures at each day whenever the total treatment exceeded 24 h. Wild-type NBD or scrambled NBD peptides (30) were dissolved in DMSO (ICN Biochemicals, Costa Mesa, CA) and added to the media at a final concentration of 50–200 µM as indicated. Equal amounts of DMSO were added as solvent control.

Immunoblot Analysis
Cells were lysed in EBC (50 mM Tris-HCl, 150 mM NaCl, 1% NP40) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µl/ml aprotinin and leupeptin) and phosphatase inhibitors (2 mM NaF and 0.5 mM sodium orthovanadate) for 30 min at 4°C. After centrifugation (4°C, 16,000xg, 10 min), protein concentrations were measured using the Bradford method (Bio-Rad, Hercules, CA). Samples containing 100 µg of protein were subjected to SDS-PAGE and transferred to a PVDF membrane (Immobilon P, Millipore Corp., Bedford, MA) for immunoblot analysis. The antibodies used were as follows: p21Cip1/Waf1 (Oncogene Research Products, Boston, MA, Ab-1); phospho-I-{kappa}B (New England Biolabs, Beverly, MA, 9241S); phospho-p38 (New England Biolabs, 9211S); and phospho-JNK/SAPK (New England Biolabs, 9251S). Antigen-antibody complexes were detected using ECL (Amersham Pharmacia Biotech, Piscataway, NJ).

Cell Cycle Analysis
Cells were trypsinized and stained with propidium iodide (Sigma), as described previously (64). Samples were analyzed by FACS using Cell Quest software (Becton Dickinson, San Jose, CA).

Analysis of Apoptosis
Cells were methanol fixed at the times indicated and stained with 1 µg/ml of bisbenzimide (Hoechst 33258; Sigma) to visualize apoptotic nuclei by fluorescence microscopy as described previously (65). Cells were scored as apoptotic based on nuclear morphology and quantified as a percentage of total cells. Five hundred cells were counted per sample and samples were blinded.

Luciferase Assay
Cells in six-well plates were transfected with 2 µg of the p21Cip1/Waf1 promoter-driven firefly luciferase reporter plasmid pWpsLuc that contains a 2.4-kb fragment of the upstream regulatory region of the human p21Cip1/Waf1 cloned into the promoterless vector pBL (66). Control cells were transfected with pBL. To allow normalization of transfection efficiencies between different plates, each transfection also contained 0.5 µg of a thymidine kinase promoter-driven renilla luciferase reporter plasmid (pRL-TK). Cells were also co-transfected with 6 µg of {Delta}N-I-{kappa}B (67) or DN-TRADD (68) or treated with 200 mM NBD wild-type or scrambled peptide and then incubated in 10 ng/ml TNF{alpha} for 12 or 24 h. Cells were lysed in 30 ml lysis buffer (Dual-Luciferase Reporter Kit, Promega, Madison, WI) per well, scraped off the plate, and centrifuged for 10 min at 4°C at 16,000xg. The supernatants were subjected to the dual luciferase assay with the firefly luciferase activity normalized by renilla luciferase expression.

RNA Interference
Double-stranded RNA oligonucleotides (Dharmacon Research, Lafayette, CO) were designed according to the manufacturer's recommendations with sequence homology to the p21Cip1/Waf1 gene, 108 nucleotides from the start codon. The sequences used were 5'-AACUUCGACUUUGUCACCGAG-3' for the wild-type complementary strand and 5'-AAGCCACUGUUUCAGCUUCGA-3' for the scrambled control. HFKs were transfected with either wild-type or control RNA duplexes using Oligofectamine (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Briefly, two solutions were prepared for each transfection: 60 pM of wild-type or scrambled double-stranded oligonucleotide in 50 µl of KSFM and 3 µl of oligofectamine in 12 µl of KSFM. The solutions were incubated at room temperature for 10 min, mixed and adjusted to a final volume of 100 µl with KSFM. The solution was evenly added to subconfluent HFKs grown on glass coverslips in 500 µl KSFM in 24-well plates. Cells were incubated with the transfection mixture overnight at 37°C and 7% CO2 before treatment with the indicated cytokines.

Immunofluorescence
HFKs were fixed to glass coverslips with 4% paraformaldehyde at the times indicated and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Coverslips were incubated overnight at 4°C in p21Cip1/Waf1 antibody (1:10 in PBS) (Oncogene Research, Ab-1). Coverslips were then rinsed in PBS and incubated for 1 h at 37°C with rhodamine red-conjugated donkey anti-mouse secondary antibody (1:100 in PBS) (Jackson Immunoresearch, West Grove, PA). The cells were rinsed in PBS, counterstained with 1 µg/ml of bisbenzimide (Hoechst 33258; Sigma) for 10 min at room temperature, and subjected to fluorescence microscopy.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Lily Yeh and David Alan Thompson for initial help with keratinocyte cultures. Special thanks to Michael May (Yale University School of Medicine, New Haven, CT) for generously providing the NBD wild-type and scrambled peptides, Michael Hinz (MDC Berlin-Buch, Germany) for the {Delta}N-I-{kappa}B construct, Dr. Jens Hasskarl for the p21Cip1/Waf1-luciferase constructs and Roya Khosravi-Far and her lab (Harvard Medical School, Boston, MA) for supplying the pMIG vector and helpful advice with this system.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 R01 CA81135 (K.M.), K16DE00275 (J.R.B.), DAAD Doktorandenstipendium im Rahmen des gemeinsamen HSP III von Bund und Ländern (A.E.) and a Senior Postdoctoral Fellowship from the New England Division of the American Cancer Society (V.Z.). Back

2 Current address: NIH/NIDCR/OPCB, Bethesda, MD. Back

Received August 8, 2002; revised December 5, 2002; accepted December 23, 2002.


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

  1. Piguet, P. F. TNF and the pathology of the skin. Res. Immunol., 144: 320–326, 1993.[Medline]
  2. Kock, A., Schwarz, T., Kirnbauer, R., Urbanski, A., Perry, P., Ansel, J. C., and Luger, T. A. Human keratinocytes are a source for tumor necrosis factor {alpha}: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. J. Exp. Med., 172: 1609–1614, 1990.[Abstract/Free Full Text]
  3. Kondo, S. and Sauder, D. N. Tumor necrosis factor (TNF) receptor type 1 (p55) is a main mediator for TNF-{alpha}-induced skin inflammation. Eur. J. Immunol., 27: 1713–1718, 1997.[Medline]
  4. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA, 72: 3666–3670, 1975.[Abstract/Free Full Text]
  5. Beutler, B. and Cerami, A. Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature, 320: 584–588, 1986.[Medline]
  6. Van Antwerp, D. J., Martin, S. J., Verma, I. M., and Green, D. R. Inhibition of TNF-induced apoptosis by NF-{kappa}B. Trends Cell Biol., 8: 107–111, 1998.[Medline]
  7. Tartaglia, L. A. and Goeddel, D. V. Two TNF receptors. Immunol. Today, 13: 151–153, 1992.[Medline]
  8. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. A novel domain within the 55 kd TNF receptor signals cell death. Cell, 74: 845–853, 1993.[Medline]
  9. Wallach, D., Boldin, M. P., Kovalenko, A. V., Malinin, N. L., Mett, I. L., and Camonis, J. H. The yeast two-hybrid screening technique and its use in the study of protein-protein interactions in apoptosis. Curr. Opin. Immunol., 10: 131–136, 1998.[Medline]
  10. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell, 84: 299–308, 1996.[Medline]
  11. Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A., Wong, W. W., and Yuan, J. Human ICE/CED-3 protease nomenclature. Cell, 87: 171, 1996.[Medline]
  12. Ashkenazi, A. and Dixit, V. M. Death receptors: signaling and modulation. Science, 281: 1305–1308, 1998.[Abstract/Free Full Text]
  13. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation [see comments]. Science, 278: 860–866, 1997.[Abstract/Free Full Text]
  14. Karin, M. and Delhase, M. The I{kappa}B kinase (IKK) and NF-{kappa}B: key elements of proinflammatory signalling. Semin. Immunol., 12: 85–98, 2000.[Medline]
  15. Li, X. H., Fang, X., and Gaynor, R. B. Role of IKK{gamma}/nemo in assembly of the I{kappa}B kinase complex. J. Biol. Chem., 276: 4494–4500, 2001.[Abstract/Free Full Text]
  16. Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. Identification and characterization of an I{kappa}B kinase. Cell, 90: 373–383, 1997.[Medline]
  17. Yaron, A., Gonen, H., Alkalay, I., Hatzubai, A., Jung, S., Beyth, S., Mercurio, F., Manning, A. M., Ciechanover, A., and Ben-Neriah, Y. Inhibition of NF-{kappa}-B cellular function via specific targeting of the I-{kappa}-B-ubiquitin ligase. EMBO J., 16: 6486–6494, 1997.[Medline]
  18. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. I{kappa}B kinase-ß: NF-{kappa}B activation and complex formation with I{kappa}B kinase-{alpha} and NIK [see comments]. Science, 278: 866–869, 1997.[Abstract/Free Full Text]
  19. Shu, H. B., Takeuchi, M., and Goeddel, D. V. The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc. Natl. Acad. Sci. USA, 93: 13973–13978, 1996.[Abstract/Free Full Text]
  20. Baker, S. J. and Reddy, E. P. Modulation of life and death by the TNF receptor superfamily. Oncogene, 17: 3261–3270, 1998.[Medline]
  21. Locksley, R. M., Killeen, N., and Lenardo, M. J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell, 104: 487–501, 2001.[Medline]
  22. Grell, M., Douni, E., Wajant, H., Lohden, M., Clauss, M., Maxeiner, B., Georgopoulos, S., Lesslauer, W., Kollias, G., and Pfizenmaier, K. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell, 83: 793–802, 1995.[Medline]
  23. Basile, J. R., Zacny, V., and Munger, K. The cytokines tumor necrosis factor-{alpha} (TNF-{alpha}) and TNF-related apoptosis-inducing ligand differentially modulate proliferation and apoptotic pathways in human keratinocytes expressing the human papillomavirus-16 E7 oncoprotein. J. Biol. Chem., 276: 22522–22528, 2001.[Abstract/Free Full Text]
  24. Symington, F. W. Lymphotoxin, tumor necrosis factor, and {gamma} interferon are cytostatic for normal human keratinocytes. J. Invest. Dermatol., 92: 798–805, 1989.[Medline]
  25. Detmar, M. and Orfanos, C. E. Tumor necrosis factor-{alpha} inhibits cell proliferation and induces class II antigens and cell adhesion molecules in cultured normal human keratinocytes in vitro. Arch. Dermatol. Res., 282: 238–245, 1990.[Medline]
  26. Pillai, S., Bikle, D. D., Eessalu, T. E., Aggarwal, B. B., and Elias, P. M. Binding and biological effects of tumor necrosis factor {alpha} on cultured human neonatal foreskin keratinocytes. J. Clin. Invest., 83: 816–821, 1989.
  27. Kono, T., Tanii, T., Furukawa, M., Mizuno, N., Taniguchi, S., Ishii, M., and Hamada, T. Effects of human recombinant tumor necrosis factor-{alpha} (TNF-{alpha}) on the proliferative potential of human keratinocytes cultured in serum-free medium. J. Dermatol., 17: 409–413, 1990.[Medline]
  28. Delvenne, P., al-Saleh, W., Gilles, C., Thiry, A., and Boniver, J. Inhibition of growth of normal and human papillomavirus-transformed keratinocytes in monolayer and organotypic cultures by interferon-{gamma} and tumor necrosis factor-{alpha}. Am. J. Pathol., 146: 589–598, 1995.[Abstract]
  29. Wang, C. Y., Mayo, M. W., and Baldwin, A. S. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B [see comments]. Science, 274: 784–787, 1996.[Abstract/Free Full Text]
  30. May, M. J., D'Acquisto, F., Madge, L. A., Glockner, J., Pober, J. S., and Ghosh, S. Selective inhibition of NF-{kappa}B activation by a peptide that blocks the interaction of NEMO with the I{kappa}B kinase complex. Science, 289: 1550–1554, 2000.[Abstract/Free Full Text]
  31. Itoh, N. and Nagata, S. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem., 268: 10932–10937, 1993.[Abstract/Free Full Text]
  32. Nagata, S. Apoptosis by death factor. Cell, 88: 355–365, 1997.[Medline]
  33. Griffith, T. S., Chin, W. A., Jackson, G. C., Lynch, D. H., and Kubin, M. Z. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J. Immunol., 161: 2833–2840, 1998.[Abstract/Free Full Text]
  34. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. Inhibition of death receptor signals by cellular FLIP [see comments]. Nature, 388: 190–195, 1997.[Medline]
  35. Kajino, S., Suganuma, M., Teranishi, F., Takahashi, N., Tetsuka, T., Ohara, H., Itoh, M., and Okamoto, T. Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-{kappa}B. Oncogene, 19: 2233–2239, 2000.[Medline]
  36. Wang, J. and Walsh, K. Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science, 273: 359–361, 1996.[Abstract]
  37. Chaturvedi, V., Qin, J. Z., Denning, M. F., Choubey, D., Diaz, M. O., and Nickoloff, B. J. Apoptosis in proliferating, senescent, and immortalized keratinocytes. J. Biol. Chem., 274: 23358–23367, 1999.[Abstract/Free Full Text]
  38. Jiang, Y. and Porter, A. G. Prevention of tumor necrosis factor (TNF)-mediated induction of p21WAF1/CIP1 sensitizes MCF-7 carcinoma cells to TNF-induced apoptosis. Biochem. Biophys. Res. Commun., 245: 691–697, 1998.[Medline]
  39. Zhang, Y., Fujita, N., and Tsuruo, T. Caspase-mediated cleavage of p21Waf1/Cip1 converts cancer cells from growth arrest to undergoing apoptosis. Oncogene, 18: 1131–1138, 1999.[Medline]
  40. Smith, M. L., Chen, I. T., Zhan, Q., O'Connor, P. M., and Fornace, A. J., Jr. Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene, 10: 1053–1059, 1995.[Medline]
  41. Seitz, C. S., Lin, Q., Deng, H., and Khavari, P. A. Alterations in NF-{kappa}B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-{kappa}B. Proc. Natl. Acad. Sci. USA, 95: 2307–2312, 1998.[Abstract/Free Full Text]
  42. Hu, Y., Baud, V., Oga, T., Kim, K. I., Yoshida, K., and Karin, M. IKK{alpha} controls formation of the epidermis independently of NF-{kappa}B. Nature, 410: 710–714, 2001.[Medline]
  43. Takeda, K., Takeuchi, O., Tsujimura, T., Itami, S., Adachi, O., Kawai, T., Sanjo, H., Yoshikawa, K., Terada, N., and Akira, S. Limb and skin abnormalities in mice lacking IKK{alpha} [see comments]. Science, 284: 313–316, 1999.[Abstract/Free Full Text]
  44. Hu, Y., Baud, V., Delhase, M., Zhang, P., Deerinck, T., Ellisman, M., Johnson, R., and Karin, M. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK{alpha} subunit of I{kappa}B kinase [see comments]. Science, 284: 316–320, 1999.[Abstract/Free Full Text]
  45. Bash, J., Zong, W. X., and Gelinas, C. c-Rel arrests the proliferation of HeLa cells and affects critical regulators of the G1/S-phase transition. Mol. Cell. Biol., 17: 6526–6536, 1997.[Abstract]
  46. Seitz, C. S., Deng, H., Hinata, K., Lin, Q., and Khavari, P. A. Nuclear factor {kappa}B subunits induce epithelial cell growth arrest. Cancer Res., 60: 4085–4092, 2000.[Abstract/Free Full Text]
  47. Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., and Chow, L. T. Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes Dev., 9: 2335–2349, 1995.[Abstract/Free Full Text]
  48. Spitkovsky, D., Hehner, S. P., Hofmann, T. G., Moller, A., and Schmitz, M. L. The human papillomavirus oncoprotein E7 attenuates NF-{kappa}B activation by targeting the I{kappa}B kinase complex. J. Biol. Chem., 277: 25576–25582, 2002.[Abstract/Free Full Text]
  49. Thompson, D. A., Zacny, V., Belinsky, G. S., Clason, M., Jones, D. L., Schlegel, R., and Münger, K. The HPV E7 oncoprotein E7 inhibits tumor necrosis factor {alpha}-mediated apoptosis in normal human fibroblasts. Oncogene, 20: 3629–3640, 2001.[Medline]
  50. Javelaud, D., Wietzerbin, J., Delattre, O., and Besancon, F. Induction of p21Waf1/Cip1 by TNF{alpha} requires NF-{kappa}B activity and antagonizes apoptosis in Ewing tumor cells. Oncogene, 19: 61–68, 2000.[Medline]
  51. Pennington, K. N., Taylor, J. A., Bren, G. D., and Paya, C. V. I{kappa}B kinase-dependent chronic activation of NF-{kappa}B is necessary for p21(WAF1/Cip1) inhibition of differentiation-induced apoptosis of monocytes. Mol. Cell. Biol., 21: 1930–1941, 2001.[Abstract/Free Full Text]
  52. el-Deiry, W. S., Tokino, T., Waldman, T., Oliner, J. D., Velculescu, V. E., Burrell, M., Hill, D. E., Healy, E., Rees, J. L., and Hamilton, S. R. Topological control of p21WAF1/CIP1 expression in normal and neoplastic tissues. Cancer Res., 55: 2910–2919, 1995.[Abstract/Free Full Text]
  53. Bren, G. D., Solan, N. J., Miyoshi, H., Pennington, K. N., Pobst, L. J., and Paya, C. V. Transcription of the RelB gene is regulated by NF-{kappa}B. Oncogene, 20: 7722–7733, 2001.[Medline]
  54. Jones, D. L., Alani, R. M., and Munger, K. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev., 11: 2101–2111, 1997.[Abstract/Free Full Text]
  55. Alani, R. M., Hasskarl, J., and Munger, K. Alterations in cyclin-dependent kinase 2 function during differentiation of primary human keratinocytes. Mol. Carcinog., 23: 226–233, 1998.[Medline]
  56. Missero, C., Calautti, E., Eckner, R., Chin, J., Tsai, L. H., Livingston, D. M., and Dotto, G. P. Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation. Proc. Natl. Acad. Sci. USA, 92: 5451–5455, 1995.[Abstract/Free Full Text]
  57. Di Cunto, F., Topley, G., Calautti, E., Hsiao, J., Ong, L., Seth, P. K., and Dotto, G. P. Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control [see comments]. Science, 280: 1069–1072, 1998.[Abstract/Free Full Text]
  58. Suzuki, A., Tsutomi, Y., Akahane, K., Araki, T., and Miura, M. Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene, 17: 931–939, 1998.[Medline]
  59. Kreuz, S., Siegmund, D., Scheurich, P., and Wajant, H. NF-{kappa}B inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol. Cell. Biol., 21: 3964–3973, 2001.[Abstract/Free Full Text]
  60. Wajant, H., Haas, E., Schwenzer, R., Muhlenbeck, F., Kreuz, S., Schubert, G., Grell, M., Smith, C., and Scheurich, P. Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD). J. Biol. Chem., 275: 24357–24366, 2000.[Abstract/Free Full Text]
  61. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, A. S., Jr. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 281: 1680–1683, 1998.[Abstract/Free Full Text]
  62. You, Z., Ouyang, H., Lopatin, D., Polver, P. J., and Wang, C. Y. Nuclear factor-{kappa}B-inducible death effector domain-containing protein suppresses tumor necrosis factor-mediated apoptosis by inhibiting caspase-8 activity. J. Biol. Chem., 276: 26398–26404, 2001.[Abstract/Free Full Text]
  63. Rheinwald, J. G. and Beckett, M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res., 41: 1657–1663, 1981.[Abstract/Free Full Text]
  64. Thompson, D. A., Belinsky, G., Chang, T. H-T., Jones, D. L., Schlegel, R., and Münger, K. The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints. Oncogene, 15: 3025–3036, 1997.[Medline]
  65. Jones, D. L., Thompson, D. A., and Munger, K. Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis. Virology, 239: 97–107, 1997.[Medline]
  66. Hasskarl, J. Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-ß1. J. Mol. Med., 78: 94–101, 2000.[Medline]
  67. Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C., and Strauss, M. NF-{kappa}B function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol., 19: 2690–2698, 1999.[Abstract/Free Full Text]
  68. Park, Y. C. A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction. Cell, 101: 777–787, 2000.[Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
J. Duan, J. Friedman, L. Nottingham, Z. Chen, G. Ara, and C. Van Waes
Nuclear factor-{kappa}B p65 small interfering RNA or proteasome inhibitor bortezomib sensitizes head and neck squamous cell carcinomas to classic histone deacetylase inhibitors and novel histone deacetylase inhibitor PXD101
Mol. Cancer Ther., January 1, 2007; 6(1): 37 - 50.
[Abstract] [Full Text] [PDF]