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RESEARCH REPORT |
B Inhibitor
1 Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and Materials Science,
2 Program in Oral Health Science, School of Dentistry, and
3 Program in Cellular and Molecular Biology, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078;
*corresponding author, cunywang{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B), which has been found to be associated with SCC development, plays an essential role in the suppression of tumor necrosis factor (TNF)-mediated apoptosis. Here, we report that an adenovirus-mediated gene transfer of NF-B inhibitor, super-repressor I kappa B alpha (Adv-SR-IB
), blocked TNF-induced NF-B activation and sensitized oral SCC cells to TNF killing. Additionally, we found that the inhibition of NFB by Adv-SR-IB enhanced TNF-mediated caspase-8 and -3 activation. These results suggest that NF-B activation is a general mechanism by which oral squamous carcinoma cells are resistant to TNF killing and provide a molecular basis for gene therapy of oral cancer by IB gene transfer in vivo.
KEY WORDS: NF-kB • gene therapy • tumor necrosis factor • apoptosis • oral squamous cell carcinoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B and the caspase cascade. Several key biological functions of TNF are displayed through those two signal pathways (Liu et al., 1996). Constitutive expression of TNF has been found in various types of human cancers, including oral SCC. Oral SCC constitutes the majority of malignancies in the oral cavity. At the late stage of malignancy, oral SCC is very resistant to cancer-therapy-mediated apoptosis (Wong et al., 1996; Dong et al., 2001). Apoptosis is programmed cell death, which is characterized by caspase cleavage, condensation of the nucleus, and DNA fragmentation. It appears to be the primary mechanism whereby cancer therapies induce the killing of tumor cells (Wang et al., 1999a, b).
NFB is a transcription factor that regulates expression of genes involved in immune responses, cell proliferation, and cell survival. NFB is a dimer or heterodimer composed of p50, p65 (Rel A), c-Rel, p52, and Rel B. In most unstimulated cells, NF-B is retained in the cytoplasm by IB-inhibitory family proteins (Baldwin, 1996). Bacterial lipopolysaccharide, viral infection, and pro-inflammatory cytokines such as TNF activate IB kinase complex (IKK) to phosphorylate the N-terminal region of IB at Ser 32 and 36. The phosphorylated IB is ubiquitinated and subsequently degraded by the 26S proteasome pathway. This liberates NF-B to translocate to the nucleus, where it activates the transcription of NF-B target genes. These genes include pro-inflammatory cytokines, chemokines, and cell survival genes (Wang et al., 1999b; Guttridge et al., 2000). Importantly, activation of NF-B and the survival genes it transcribes has been found to be a key mechanism by which cells are resistant to TNF-mediated apoptosis (Beg and Baltimore, 1996; Van Antwerp et al., 1996; Wang et al., 1996, 1998, 1999b; Mayo et al., 1997). Inhibition of NF-B activation by multiple approaches has been shown to sensitize cells to TNF-mediated apoptosis (Wang et al., 1999b).
Recently, NF-B has been found to be associated with the development and progression of several human malignancies, including head and neck cancer, pancreatic cancer, and breast cancer (Duffey et al., 1999; Dong et al., 2001). Several studies have implicated the NF-B signaling pathway as being associated with the progression and metastasis of SCC (Huang et al., 2000; Alevizos et al., 2001; Dong et al., 2001). Currently, most approaches to the inhibition of NF-B are via stable or transient transfection of the modified form of IB, SR-IB (Wang et al., 1998, 1999a; You et al., 2001). SR-IB contains two serine to alanine substitutions at residues 32 and 36, which renders it resistant to phosphorylation by the IKK complex and subsequent ubiquitination and degradation. Thereby, SR-IB inhibits NF-B activation by blocking the nuclear translocation of NF-B (Wang et al., 1996; Mayo et al., 1997; Guttridge et al., 2000).
In this report, we explore whether inhibition of NF-B by gene therapy approaches (Shillitoe, 1998; Gibson et al., 2000; Zhu et al., 2001) can sensitize oral SCC cells to TNF-mediated apoptosis. We found that recombinant adenoviruses effectively transduce SCC cells and deliver SR-IB proteins to block TNF-mediated NF-B activation. Whereas the parental SCC cells were resistant to TNF killing, SR-IB-transduced cells were sensitive to TNF killing. In addition, we found that the caspase cascade was activated by TNF under inhibition of NF-B by SR-IB gene transfer. These results provide a molecular basis for gene therapy of oral cancer in vivo by SR-IB gene transfer.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Adenoviral Infection
Adenoviruses expressing SR-IB or adenoviruses expressing ß-galactosidase (Adv-LacZ) were produced as described previously (Wang et al., 1999a). For the determination of viral transduction efficiency, 1 x 105 cells were plated in six-well plates for 24 hrs. Cells were infected with Adv-LacZ at a variety of multiplicities of infections (MOIs) from 10 to 500. Four hours after infection, the virus-containing medium was aspirated and replaced with normal growth medium for 24 hrs. Cells were then washed with PBS, fixed with 0.5% glutaraldehyde, and stained with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal). The percentage of positive cells was counted with a hematocytometer. We found that about 90% of cells could be infected by Adv-LacZ at an MOI of 200 with minimal cell cytotoxicity. Thus, an MOI of 200 was chosen for viral infection in all experiments performed in our studies. For cell-killing assay, cells were infected with Adv-SR-IB or Adv-LacZ at an MOI of 200 for 4 hrs. Twenty-four hrs later, cells were treated with TNF for an additional 24 hrs. Cell viability was determined with a trypan blue exclusion assay.
Cell Death Enzyme-linked Immunosorbent Assay (ELISA)
For cell death ELISA, supernatants were collected from both TNF-stimulated and unstimulated cells and frozen in –70°C. Twenty-µL aliquots of supernatant were used for the assessment of DNA fragmentation and histone release from the nucleus. The assays were performed according to the manufacturer's protocol (Roche, Mannheim, Germany).
Western Blot Analysis
Whole-cell extracts were prepared as described previously (Wang et al., 1999b). The extracts were subjected to sodium dodecyl sulfate, 10% polyacrylamide gel electrophoresis, and transferred to PVDF membrane by a semi-dry transfer apparatus (BioRad, Hercules, CA, USA). Proteins were probed with primary antibodies and visualized by means of an ECL kit (Amersham, Piscataway, NJ, USA) according to the manufacturer's instruction. For internal control, the blots were stripped with 62.5 mM Tris buffer (pH 6.8) containing 100 mM 2-mercaptoethanol and 2% SDS at 60°C for 1 hr and re-probed for -tubulin (Wang et al., 1999b). Primary antibodies were from the following sources: monoclonal antibodies against human IB (1:1000) (Santa Cruz, CA, USA); polyclonal antibodies against phospho-specific IB (1:1000) (Cell Signaling, Beverly, MA, USA); and monoclonal antibody against -tubulin (1:5000) (Sigma, St. Louis, MO, USA).
In vitro Caspase-3 and –8 Assay
Both viral infection and TNF treatment were performed as described above for the Western blot analysis. Caspase-3 and –8 activities were determined with a CaspACE assay kit from Promega (Madison, WI, USA) and ApoAlert caspase-8 colorimetric assay kit from Clonetech (Palo Alto, CA, USA), respectively. Briefly, the detached and attached cells were collected, washed with PBS, and lysed in 200 µL of ice-cold cell lysis buffer provided by the manufacturers. The cell extracts were centrifuged, and supernatants were collected. From 200 to 300 µg of protein extracts were incubated in reaction buffer containing IETD-pNA (colorimetric caspase-8 substrate) or DEVD-pNA (colorimetric caspase-3 substrate) at 37°C for 2 to 3 hrs. The samples were analyzed with a plate reader by the measurement of optical density (OD) at a wavelength of 405 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B signaling, we have produced replication-defective adenoviruses expressing SR-IB (Wang et al., 1999a). To determine whether Adv-SR-IB inhibited NF-B activation in oral SCC cells, we infected SCC15 cells with Adv-SR-IB or Adv-LacZ (control virus). As shown in Fig. 1A
, SR-IB is highly expressed in SCC15 cells, as determined by Western blot analysis. Of note, the molecular weight of SR-IB was slightly larger than that of endogenous IB because of a Flag-tagged epitope. Due to the fact that the promoter of IB itself is transcriptionally controlled by NF-B, ectopic expression of SR-IB inhibited endogenous expression of IB (lane 1 compared with lane 5). For further determination of whether SR-IB functioned to block the phosphorylation and degradation of IB, cells were treated with TNF. As shown in Fig. 1B, TNF induced phosphorylation and degradation of IB in cells infected with control virus. The level of phosphorylated IB in cells infected with Adv-LacZ was gradually reduced following TNF stimulation because of its degradation by the 26S proteasome pathway. In contrast, the level of SR-IB remained unchanged. Consistently, SR-IB also inhibited TNF-induced nuclear translocation of NF-B and NF-B transcriptional activity (You et al., 2001).
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B by Adv-SR-IB would render cells sensitive to TNF killing. As shown in Fig. 2A, cells infected with control viruse Adv-LacZ were resistant to TNF killing. In contrast, cells infected with Adv-SR-IB were rounded, detached from plates, and floated in the medium following TNF stimulation. The trypan blue exclusion assay found that over 50% of cells infected with Adv-SR-IB, but not cells infected with Adv-LacZ, were killed 24 hrs after TNF treatment (Fig. 2B). At the late stage of apoptosis, the cell membrane is disrupted, and genomic DNA is cleaved into fragments by specific DNA fragmentation factors (Ashkenazi and Dixit, 1998). Fragmented DNA along with histone protein is released into the medium. To confirm that the SR-IB gene-transfer-enhanced TNF killing was by an apoptotic mechanism, we also performed cell death ELISA to measure DNA fragmentation. As shown in Fig. 2C, following TNF treatment, significant DNA fragments were detected in cells infected with Adv-SR-IB, but not in cells infected with Adv-LacZ. Additionally, we also performed experiments to determine whether Adv-SR-IB also rendered other oral SCC cells sensitive to TNF killing. As shown in Fig. 3, SCC5 cells infected with Adv-SR-IB were more sensitive to TNF killing than were cells infected with Adv-LacZ.
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B blocked caspase-8 activation in human fibrosarcoma cells (Wang et al., 1998), we also tested whether TNF could activate caspase-8 in SCC cells after Adv-SR-IB transduction. To measure caspase-8 activity, we prepared whole-cell protein extracts and incubated them with specific caspase-8 substrate IETD-pNA. As shown in Fig. 4A, caspase-8 activity was strongly induced in cells infected with Adv-SR-IB but not in cells infected with Adv-LacZ 8 hrs after TNF treatment. Caspase-8 activity slowly decreased 16 hrs after TNF treatment. The active caspase-8 can directly or indirectly activate the executing caspase-3 to cleave specific cellular proteins leading to cell death. As shown in Fig. 4B, caspase-3 was activated in cells infected with Adv-SR-IB but not with Adv-LacZ following TNF treatment. Activation of caspase-3 was relatively slower than that of caspase-8, indicating that caspase-8 is an initiating caspase in SCC cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B and TNF was efficient in the induction of apoptosis of oral SCC cells. To the best of our knowledge, this is the first demonstration that inhibition of NF-B by IB gene transfer sensitizes oral SCC cells to TNF killing.
Our studies demonstrate that TNF can initiate the caspase cascade in oral SCC cells under inhibition of NF-B. The results suggest that components of cell death machinery are intact in oral SCC cells. Currently, it is unknown how NF-B suppresses TNF-mediated apoptosis in oral SCC cells. Using a human fibrosarcoma cell model system, we have identified several NF-B-regulated anti-apoptotic genes. These genes include inhibitors of apoptosis family proteins, TNFR-associated factor family proteins, Bcl-2 family members A1 and Bcl-XL, IEX-1L and recently cloned NDED (Wang et al., 1998, 1999b; You et al., 2001). NDED was found to be induced in oral SCC cells by TNF through activation of NF-B (You et al., 2001). It will be interesting to determine whether other NF-B-regulated anti-apoptotic genes are induced by TNF in oral SCC cells.
Aberrant activation of NF-B has been implicated in the development and progression of human cancers, including head and neck squamous cell carcinoma (Duffey et al., 1999; Dong et al., 2001). Several pro-inflammatory cytokines and pro-angiogenic factors that are regulated by NF-B have been found to be up-regulated in SCC cells (Duffey et al., 1999; Dong et al., 2001). Interestingly, NF-B-regulated gene products were also found to be associated with oral cancer metastasis (Dong et al., 2001). Since ectopic expression of IB in human head and neck SCC cells inhibits tumor growth in vivo (Duffey et al., 1999), the strategy for oral cancer gene therapy by inhibition of NF-B may have a dual function: promotion of apoptosis and suppression of tumorigenesis. However, given the ubiquitous presence of NF-B and TNF receptors, non-specific inhibition of NF-B function may have cytotoxic affects on normal tissue. The challenge of the future will be to develop a specific gene transfer system that can selectively deliver IB into tumor cells but not into normal cells. Nevertheless, our results provide an important framework for the examination of whether inhibition of NF-B will enhance cancer therapy in vivo.
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ACKNOWLEDGMENTS |
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Received August 28, 2001; Last revision December 14, 2001; Accepted December 18, 2001
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Ashkenazi A, Dixit V 1998). Death receptors: signaling and modulation. Science 281:1305–1308.
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