Title: Rb-independent Induction of Apoptosis by Bovine Papillomavirus Type 1 E7 in Response to Tumor Necrosis Factor α
Abstract: Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor α (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A2 was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB. Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor α (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A2 was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB. bovine papillomavirus type 1 human papillomavirus tumor necrosis factor α retinoblastoma protein glutathioneS-transferase embryonic fibroblasts propidium iodide arachidonic acid cytosolic phospholipase A2 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide enzyme-linked immunosorbent assay phosphate-buffered saline polymerase chain reaction Papillomaviruses are small DNA viruses that infect various epithelial tissues, including the epidermis and the epithelial linings of the anogenital tract. Papillomaviruses replicate in the stratified layers of skin and mucosa and usually give rise to benign lesions such as warts or papillomas. Some animal papillomaviruses, including bovine papillomavirus type 1 (BPV-1),1 induce fibropapillomas. Because of its ability to transform cells and to replicate its genome in established murine cell lines, BPV-1 has served as the prototype for studies of molecular biology of the papillomaviruses (for review see Ref. 1Howley P.M. Fields B.N. Knipe D.M. Howley P.M. 3rd Ed. Fields Virology. 2. Lippincott-Raven, Philadelphia, PA1996: 2045-2076Google Scholar). Specific types (“high risk”) of human papillomaviruses (HPV) infect the anogenital tract and are strongly associated with the development of cervical carcinoma (for review see Ref. 2zur Hausen H. Biochim. Biophys. Acta. 1996; 1288: F55-F78Crossref PubMed Scopus (1489) Google Scholar). The low risk HPV types, such as 6 and 11, are found associated primarily with benign lesions that rarely progress to cancer. Papillomavirus oncogenes manifest their transforming potential in various cell culture based assays and transgenic models (1Howley P.M. Fields B.N. Knipe D.M. Howley P.M. 3rd Ed. Fields Virology. 2. 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In the present study, we investigated the biological and biochemical activities of BPV-1 E7. In particular, the susceptibility of cells expressing BPV-1 E7 in response to tumor necrosis factor α (TNF) treatment and the role of Rb in this process were examined. Our results indicated a Rb-independent mechanism of apoptosis by BPV-1 E7. The retrovirus vector pBabe Puro is a Moloney murine leukemia virus-based vector containing a puromycin resistance gene (51Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1903) Google Scholar). Plasmids pBE7 and pBAUE7 encode BPV-1 E7 and the AU1 epitope-tagged-E7 fusion in pBabe Puro, respectively. Plasmids encoding HPV-6b E7 and HPV-16 E7 were described (4Munger K. Werness B.A. Dyson N. Phelps W.C. Harlow E. Howley P.M. EMBO J. 1989; 8: 4099-4105Crossref PubMed Scopus (927) Google Scholar, 52Barbosa M.S. Edmonds C. Fisher C. Schiller J.T. Lowy D.R. Vousden K.H. 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Primary Rb+/+ and Rb−/− mouse embryonic fibroblasts (MEF) were kindly provided by Dr. Tyler Jacks. To establish stable BPV-1 E7-expressing cell lines, plasmids encoding wild-type BPV-1 E7 and AU1 epitope-tagged E7 in pBabe Puro vector were transfected into the amphotrophic retrovirus packaging cell line PA317 (57Miller A.D. Buttimore C. Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1144) Google Scholar), respectively, by calcium phosphate-mediated transfection. Transfected cells were selected for puromycin resistance. Viruses were collected and titered on C127 cells to determine the puromycin-resistant colony-forming units. C127 or MEF cells were then infected with retroviruses containing an approximately equal number of colony-forming units. After puromycin selection, populations of infected cells were pooled and used for subsequent experiments. All experiments were performed using cells within 12 passages (7 passages for MEFs). Cells were seeded in 96-well plates at a density of 1000 cells/well. The following day, the medium was changed to regular medium (untreated cells) or medium supplemented with various concentrations of murine recombinant TNF (Sigma) as indicated in the text or figure legend (treated cells). In some experiments, TNF was added to the medium together with 2 μg/ml of cycloheximide as indicated in figure legend. Following treatment with TNF, viable cells were measured using the quantitative colorimetric MTT assay kit (Chemicon International Inc., Temecula, CA) according to the manufacture's protocol. MTT is cleaved by living cells to yield a dark blue formazan product. Plates were analyzed in an ELISA plate reader at 570 nm with a reference wavelength of 655 nm. Cells were seeded in 96-well plates at a density of 1000 cells/well. The following day, the medium was changed to regular medium (untreated cells) or medium containing 10 ng/ml TNF (treated cells) and incubated for 24 h. Of 200 μl of cell extract collected from each well, 20 μl were used for analysis of nucleosomes in cytoplasmic fractions by a Cell Death Detection ELISAplus kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. Enrichment factor represents the absorbance measured at 405 nm with a reference of 492 nm of treated cells divided by that of the corresponding untreated cells. Cells were seeded in 6-well plates at 2 × 105/well. The following day, the medium was changed to regular medium (untreated cells) or medium containing 1 ng/ml TNF plus 1 μg/ml cycloheximide (treated cells) and incubated for hours as indicated in figure legends. For viability assay, both floating and adherent cells were harvested and pelleted. Cells were resuspended in PBS containing 1 μg/ml of propidium iodide (PI), and the fluorescence was measured by flow cytometry on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) in logarithmic scale. For DNA fragmentation analysis, cells were processed as described previously (58Ferlini C. Biselli R. Scambia G. Fattorossi A. Cell Prolif. 1996; 29: 427-436Crossref PubMed Scopus (16) Google Scholar) with slight modifications. Briefly, both floating and adherent cells were harvested and fixed in 50% ethanol at 4 °C overnight. Following fixation, the cells were centrifuged and resuspended in 0.5 ml of sample buffer (one part of PBS and three parts of 0.2m Na2HPO4, 0.1 m citric acid, pH 7.8, 0.1% Triton X-100 with 10 μg/ml of PI). After an incubation of 30 min. at room temperature, the cell samples were stored on ice and analyzed for DNA content on the FACScan flow cytometer in linear scale. Cells were seeded in 96-well plates at a density of 1000 cells/well. After adhering to the plates, cells were labeled overnight in 200 μl of complete medium containing 0.33 μCi/ml [3H]AA ([5, 6, 8, 11, 12, 14, 15-3H]AA, 100 μCi/ml; NEN Life Science Products). The cells were then washed twice with PBS and incubated for 48 h in regular medium or medium containing 10 ng/ml of TNF. The medium was collected, and the amount of released AA was determined by liquid scintillation counting. GST fusion proteins were expressed in Escherichia coli strain DH5α. One-liter cultures were inoculated with 100 ml of stationary cultures and grown for 1 h before induction with 0.2 mmisopropyl-β-d-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation, resuspended in 50 ml of low salt association buffer (100 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1% Nonidet P-40, and 1 mmphenylmethylsulfonyl fluoride) plus 0.03% SDS, 2 mmdithiothreitol, and lysed by sonication. After centrifugation at 10,000 × g for 10 min., supernatant was collected and mixed with glutathione-Sepharose beads (Amersham Pharmacia Biotech). After rotary shaking for 2 h at 4 °C, the beads were collected by centrifugation at 1000 × g, washed three times with 20 volumes of low salt association buffer, and stored at 4 °C.In vitro translated E7 proteins were prepared by using the rabbit reticulocyte lysate transcription and translation system (Promega) and 35S-labeled cysteine (ICN Biomedicals, Irvine, CA). For in vitro binding, 30 μl of glutathione-Sepharose beads containing 2 μg of GST fusion proteins were combined with 2–20 μl of 35S-labeled in vitro translated proteins in lysis buffer (250 mm NaCl, 20 mm Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 1 mm EDTA, 2 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) in a total volume of 250 μl. The mixtures were subjected to rotary shaking for 3 h at 4 °C. The mixtures were then washed extensively with lysis buffer, boiled in SDS sample buffer, and electrophoresed on SDS-polyacrylamide gels. Gels were dried and scanned by Molecular Imager (Bio-Rad). To detect E7 proteins, proliferating cells were metabolically labeled overnight with 1 mCi of 35S-labeled cysteine/10-cm dish in cysteine-free Dulbecco's minimum essential medium containing 5% dialyzed fetal calf serum. Cells were lysed at 4 °C in 1 ml of lysis buffer. Insoluble debris was pelleted by centrifugation at 10,000 × g for 15 min, and the supernatant was incubated with anti-AU1 antibody (BAbCO) and protein A-Sepharose beads. After extensive washes with lysis buffer, the bound proteins were released from the beads by boiling in SDS sample buffer and loaded onto a 15% SDS-polyacrylamide gel. The epitope-tagged BPV-1 E7 band was analyzed by Molecular Imager. To compare p53 levels, PBE7 and PURO cells were metabolically labeled overnight with [35S]methionine. Cell extracts were prepared by lysing cells with 0.1% Nonidet P-40 lysis buffer (34Jones D.L. Thompson D.A. Munger K. Virology. 1997; 239: 97-107Crossref PubMed Scopus (223) Google Scholar). Lysates containing equal numbers of cpms were immunoprecipitated with a p53 monoclonal antibody (Ab421; Amersham Pharmacia Biotech). Following extensive wash, the bound proteins were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel and analyzed by Molecular Imager. To examine the stability of pRB, exponentially growing PBE7 and PURO cells were treated with 20 μg/ml of cycloheximide for various time periods, harvested, and lysed in the 0.1% Nonidet P-40 lysis buffer. After removing cell debris by centrifugation, 130 μg of proteins were fractionated on a 6% SDS-polyacrylamide gel. The gel was then blotted simultaneously with an anti-pRB monoclonal antibody pMG3–245 (PharMingen) and an anti-tubulin β antibody (Sigma). The antigen-antibody complexes were detected by chemiluminescence (Pierce). To detect E7 mRNA expression, 1 μg of total cellular RNA isolated from various cell lines was used as a template to synthesize cDNA using SuperScript II reverse transcriptase and an oligo(dT) primer (Life Technologies, Inc.). BPV-1 E7 specific primers (sense, nucleotides 1–19: 5′-ATGGTTCAAGGTCCAAATA-3′; antisense, nucleotides 381–365: 5′-TCGTTTGCCATGACGCT-3′) were used to amplify a 381-nucleotide fragment from the E7 cDNA. Kruskal-Wallis test has been used to assess statistical significance of differences in E7-expressing cells and control cells. p < 0.05 was considered significant. To investigate the effect of BPV-1 E7 expression on cell growth, a cell line that expresses BPV-1 E7, named PBE7, was established. For this purpose, C127 cells were infected with amphotrophic retrovirus expressing BPV-1 E7. After puromycin selection, populations of infected cells were pooled and used for subsequent experiments. To avoid the possibility of chromosomal instability because of the expression of BPV-1 E7, all experiments described here were performed using cells within 12 passages. To facilitate detection, a cell line that expresses BPV-1 E7 with a C-terminal AU1 epitope tag (PBAUE7) was also made. BPV-1 E7 gene expression was confirmed in the E7 expressing cell lines by PCR amplification of the cDNA after reverse transcription of cellular mRNA (Fig. 1 A). To examine the expression of BPV-1 E7 protein, a monoclonal antibody against the AU1 epitope was used to precipitate the epitope-tagged E7 protein from PBAUE7 cells, because antibody to BPV-1 E7 was not available. In agreement with previous observations in BPV-1-transformed cells (50Jareborg N. Alderborn A. Burnett S. J. Virol. 1992; 66: 4957-4965Crossref PubMed Google Scholar), BPV-1 E7 protein was present at a low level (Fig.1 B). This result indicates that in the PBAUE7 cells, overexpression of the E7 protein did not occur. Morphologically, PBE7 and PBAUE7 cells were indistinguishable from parental cells or retrovirus vector-infected PURO cells (59Rapp L. Liu Y. Hong Y. Androphy E.J. Chen J.J. Oncogene. 1999; 18: 607-615Crossref PubMed Scopus (12) Google Scholar). For comparison, C127 cells expressing BPV-1 E6 (PBE6) were also examined (59Rapp L. Liu Y. Hong Y. Androphy E.J. Chen J.J. Oncogene. 1999; 18: 607-615Crossref PubMed Scopus (12) Google Scholar). Within 24 h of confluence, PBE6 cells piled up and became highly spindle shaped, indicating a loss of contact inhibition as observed in focus formation assays with BPV-1 E6. Unlike PBE6 cells, cell piling-up was not found in PBE7 or PBAUE7 cells for up to 3 weeks (data not shown). This phenotype of E7-expressing cells is consistent with the results of focus formation assays, in which no foci were observed in C127 cells infected with BPV-1 E7-expressing retrovirus. In contrast, BPV-1 E6 efficiently induces focus formation in this assay. This result is also in agreement with the previous report that BPV-1 E7 transformed neither C127 nor NIH3T3 cells (60Schiller J.T. Androphy E.J. Vass W.C. Lowy D.R. Cancer Cells/DNA Tumor Viruses. 1986; 4: 571-579Google Scholar). We have previously observed that expression of BPV-1 E6 sensitizes C127 cells to TNF-induced apoptosis (59Rapp L. Liu Y. Hong Y. Androphy E.J. Chen J.J. Oncogene. 1999; 18: 607-615Crossref PubMed Scopus (12) Google Scholar). To examine the TNF susceptibility of E7-expressing cells, PBE7 and PBAUE7 cells were treated with various concentrations of murine TNF, and cell viability was determined quantitatively by analysis of MTT conversion (61Hansen M.B. Nielsen S.E. Berg K. J. Immunol. Methods. 1989; 119: 203-210Crossref PubMed Scopus (3351) Google Scholar). As shown in Fig. 2 A, E7-expressing cells were much more susceptible to TNF treatment compared with the control cells. Although TNF induced less than 4% of the PURO cells to undergo cytolysis at a concentration of 10 ng/ml, 28% of PBE7 cells exhibited cytolysis. The TNF sensitivity of PBAUE7 cells was similar to that of PBE7 cells, suggesting that the AU1 tag did not alter the activity of BPV-1 E7. The TNF sensitivity of E7-expressing cells was compared with that of PBE6 cells. We previously showed that BPV-1 E6 was a strong inducer of apoptosis compared with other known pro-apoptosis viral oncogenes such as the polyomavirus middle T antigen (59Rapp L. Liu Y. Hong Y. Androphy E.J. Chen J.J. Oncogene. 1999; 18: 607-615Crossref PubMed Scopus (12) Google Scholar). As shown in Fig.2 A, the effects of BPV-1 E6 and E7 on cell viability were similar, indicating that BPV-1 E7 is an equally potent inducer of cell death. In addition to the MTT conversion assay, we employed an alternate assay to evaluate cell viability. This assay is based on the fact that live cells with intact plasma membrane exclude PI because of the charged nature of PI, whereas dead or dying cells with damaged cell membrane uptake PI and thus fluoresce when PI intercalates into DNA. After TNF and cycloheximide treatment, both floating and adherent cells were pooled, resuspended in PBS containing a low concentration of PI, and analyzed on a FACScan flow cytometer. As shown in Fig. 2 B, whereas cell death in TNF-treated PURO cells showed modest increase over the spontaneous cell death in untreated PURO culture, TNF-treated PBE7 cells exhibited bigger increase in cell death. TNF kills most cell types by apoptosis rather than necrosis (62Laster S.M. Wood J.G. Gooding L.R. J. Immunol. 1988; 141: 2629-2634PubMed Google Scholar). Although the MTT conversion assay and the PI permeability analysis measure cell survival and cytotoxicity, they do not differentiate between cells dying of necrosis or apoptosis. To examine whether the cytolysis of PBAUE7 cells after TNF treatment is apoptotic, we performed the Cell Death Detection ELISAplusassay. This assay provides a qualitative and quantitative determination of cytoplasmic histone-associated DNA fragments resulting from DNA degradation that occurs specifically in apoptotic cells. During the process of apoptosis, a number of cellular proteases and endonucleases are activated and cellular DNA is degraded to characteristic nucleosome-sized fragments. Treatment of PBAUE7 cells with TNF for 24 h resulted in specific enrichment of mono- and oligonucleosomes released into the cytoplasm (Fig.3 A). Approximately 2.5-fold enrichment of nucleosomes in the cytoplasm was observed in PBAUE7 cells as compared with PURO cells. These results demonstrate that E7 expressing cells undergo enhanced apoptosis after TNF treatment. To assess TNF-induced DNA fragmentation further, we compared the TNF-induced DNA fragmentation in E7-expressing cells and the control cells by flow cytometric analysis. Samples from TNF-treated or untreated cells were stained with PI after fixation, and the DNA content was analyzed on a FACScan flow cytometer. Both PURO and PBE7 cells showed a sub-G1 population in response to TNF. However, PBE7 cells exhibited approximately 2.5-fold increase of sub-G1 population relative to that of PURO cells after incubation with TNF, indicating that E7 expression enhances TNF-induced DNA fragmentation (Fig. 3 B). The TNF-induced lysis of susceptible cells is usually accompanied by the release of AA into the culture medium (63Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar). The release of AA also accompanies the lysis of cells rendered sensitive to TNF by inhibitors of transcription or translation and some viral proteins (64Voelkel-Johnson C. Thorne T.E. Laster S.M. J. Immunol. 1996; 1