necrosis factor (TNF)-α (10 ng/ml) and cycloheximide (30 μg/ml) for 7 hr. AT6 cells (21), which express a temperature-sensitive p53 allele, were placed at 32°C for 3 days for the induction of apoptosis. Cells were then harvested, apoptotic cells were identified by terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay (22, 23), and expression of the E4orf6 protein was detected by immunofluorescence using the E4orf6-specific RSA3 antibody and Texas red-coupled donkey anti-mouse IgG.
Transformation Assay. Primary Fischer baby rat kidney cells were prepared as described (17). Cells were transfected by the calcium phosphate technique (24) and maintained in medium with 5% fetal calf serum. Transformed cultures were stained with Giemsa stain, and foci that were 4 mm or greater in diameter were counted at 30 days after transfection. Three independent experiments were performed for each plasmid combination. Five pCMVE1A/pCMVE4orf6 transformants, five pXhoC/MMTVE4orf6 transformants, and five pXhoC/ CMVE4orf6 transformants were cloned and maintained as continuous cell lines.
Selection of 293 Cell Lines Expressing E4orf6 Protein. The 293 human embryonic kidney cell line expresses the adenovirus E1A and E1B genes (25). The 293 cells were transfected with pMMTVE4orf6 and pMMTVE2A together with the pBabePuro puromycin-resistance marker (26) by the calcium phosphate technique. Clones were selected and maintained in medium containing 10% delipidated calf serum plus puromycin at 1 μg/ml.
Protein Analysis. For analysis of proteins by immunoprecipitation followed by Western blotting, cells were lysed in RIPA buffer (50 mM Tris·HCl, pH 7.4/150 mM NaCl/1% Triton X-100/0.1% SDS/1% sodium deoxycholate) and normalized for protein concentration, and specific proteins were immunoprecipitated with RSA3 (E4orf6-specific monoclonal antibody, ref. 27), 421 (p53-specific monoclonal antibody, ref. 28), or 2A6 (E1B 55-kDa-specific monoclonal antibody, ref. 29). The immunoprecipitates were subjected to electrophoresis in an SDS-containing polyacrylamide gel, and proteins were transferred to an Immobilon membrane (Millipore), which was then incubated with E4orf6- or p53-specific monoclonal antibody. Reactive protein species were detected by enhanced chemiluminescence (ECL; Amersham).
To assay proteins by immunofluorescence, cells were grown on coverslips. The coverslips were washed in phosphate-buffered saline and fixed in 100% methanol. The fixed calls were then incubated with an E4orf6-, E1B 55-kDa-, or p53-specific antibody followed by reaction with a secondary antibody conjugated with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine B isothiocyanate (TRITC) and were examined with a confocal microscope.
The half-life of p53 in transformed baby rat kidney cells was determined by pulse-chase analysis (30).
PCR Amplification of cDNAs. Total cell RNA was prepared from transformed baby rat kidney cells, treated with RNase-free DNase I (1 unit/μg of RNA) (GIBCO/BRL), and subjected to reverse transcription with Superscript II polymerase (GIBCO/BRL) using a 3′-E4orf6-specific primer (P3, 5′-AATCCCACACTGCAGGGA-3′). The cDNA was then amplified with KlenTaq DNA polymerase (CLONTECH) in a PCR using P3 primer and a 5′-E4orf6-specific primer (P2, 5′-CGGCGCACTCCGTACAGT-3′). To control for the presence of DNA that might have survived treatment of the RNA preparations with DNase I, a second amplification was performed on each sample with a primer from the promoter region of the plasmid used to express the E4orf6 mRNA (P1, 5′-CGGTAGGCGTGTACG-3′) and the P3 primer.
Tumor Induction. Transformed cells were injected subcutaneously into 6- to 8-week-old female Swiss 3T3 nude mice (Taconic Laboratories) and assayed by palpation until a tumor was detected. Animals that did not develop a tumor were sacrificed on day 110.
The Adenovirus E4orf6 Protein Blocks p53-Induced Apoptosis. Since the adenovirus E4orf6 protein can bind to p53 and block its ability to activate transcription (13), we tested whether the E4orf6 protein could prevent apoptosis induced by p53. We employed AT6 cells for the assay. These cells lack an endogenous p53 gene and harbor an ectopic temperature-sensitive p53 allele (21). They grow normally when maintained at the nonpermissive temperature but undergo apoptosis when p53 function is restored by shifting to the permissive temperature. AT6 cells were transfected with a plasmid expressing the E4orf6 gene under control of the cytomegalovirus immediate early promoter; 24 hr later the cultures were shifted to the permissive temperature, and the cultures were assayed for apoptosis after incubation for 72 hr at 32°C. Transfected cells expressing the E4orf6 protein were identified by immunofluorescence using an E4-specific monoclonal antibody, and apoptotic cells were identified by using the TUNEL assay (22, 23). In this assay, chromatin is treated in situ with terminal deoxynucleotidyltransferase to label 3′-OH ends of DNA with biotin-dUTP, followed by reaction with fluorescein-labeled avidin to identify cells with fragmented DNA. In the confocal micrograph displayed in Fig. 1A, E4orf6-specific immunofluorescence is represented by the red signal and DNA fragmentation is monitored with the green signal. The red nuclei from transfected cells expressing the E4orf6 protein display little yellow signal (yellow results from the overlap of green and red signals), whereas the nuclei of cells that do not express the E4orf6 protein display a green signal indicative of DNA fragmentation, a hallmark of apoptosis. Expression of the E4orf6 protein (red signal) and expression of DNA fragmentation (green signal) are mutually exclusive, indicating that the viral protein protects against p53-induced apoptosis in AT6 cells.
It is noteworthy that the green apoptotic nuclei in Fig. 1A appear to be much smaller than the red E4orf6-expressing nuclei that were protected from apoptosis. However, examination of cells by phase-contrast microscopy (data not shown) revealed that nuclei undergoing apoptosis had not shrunk to a considerable extent. Rather, the green signal indicative of DNA fragmentation was limited to subdomains within apoptotic nuclei. Presumably the nuclear shrinkage that is characteristic of apoptosis would be observed at later times after the induction of p53.
We also tested the ability of the E4or6 protein to protect against the induction of apoptosis in H1299 cells that do not express p53. H1299 cells were transfected with the E4orf6-expressing plasmid, and 24 hr later the cultures were treated for 7 hr with TNF-α (10 ng/ml) and cycloheximide (30 μg/ml) to induce apoptosis. In the confocal micrograph shown in Fig. 1B, the E4orf6 protein is identified by immunofluorescence (red signal) and DNA fragmentation is marked by the TUNEL assay (green signal). In this case, overlapping green and red signals (yellow signal) are evident, indicating that the E4orf6 protein does not protect cells from the induction of p53-independent apoptosis by TNF-α. As noted above, nuclei undergoing apoptosis have not shrunk, and the yellow signal generated by DNA fragmentation was limited to subdomains within the nuclei whose boundaries are demarcated by the E4orf6-specific immunofluorescence (red signal).
E4orf6 Cooperates with E1A and E1B to Transform Rat Cells. The E1B proteins cooperate with E1A proteins to transform rodent cells at least in part by blocking apoptosis (7). Since the E4orf6 protein, like E1B proteins, can block p53-mediated apoptosis, we explored the possibility that it might contribute to oncogenesis. Primary baby rat kidney cells were