Technical Reference #293

293.

Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways. Susanne I. Wells, Delicia A. Francis, Alla Y. Karpova, Jennifer J. Dowhanick, John D, Benson, Peter M. Howley, Harvard Medical School, Millenium Pharmaceuticals, Children's Hospital, University of Pennsylvania, Enanta Pharmaceuticals, The EMBO Journal, 19(293), 5770, (2000)
Link To Paper

Abstract:
A hallmark of human papillomavirus (HPV) associated carcinogenesis is the integration of the viral DNA into the cellular genome, usually accompanied by the loss of expression of the viral E2 gene. E2 binds to and represses the viral promoter directing exp

Keywords:
E2, p21 CIP, papillomavirus, pRB, senescence

Materials & Methods:
Recombinant plasmids Expression plasmids for BPV1 E2-TA (p2450) and E2-TR (p1153) proteins (Spalholz et al., 1991; Winokur and McBride, 1992; Dowhanick et al., 1995), for the HPV16 wild-type (p2091) or mutant I73A (p3670) and E39A (p3667) E2 proteins (Sakai et al., 1996), as well as for HPV 18 E2 (p2092) (Del Vecchio et al., 1992), have been described previously. The numbers indicated for each of these plasmids represent their respective numbers in our laboratory plasmid bank. Expression vectors for the wild-type and mutant Ad 12S E1A proteins were a generous gift from Dr T.Kouzarides (Reid et al., 1998). E1A expression is under the control of an RSV promoter in the context of plasmid pBJ9 (RSV empty vector). To construct analogous expression vectors for the HPV oncoproteins, we subcloned coding sequences for the HPV 16 E6 and E7 proteins as well as for the mutant E7 DLYC protein (Münger et al., 1989) into the pCR-Blunt II-TOPO plasmid vector using PCR and the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The respective sequences were then excised and subcloned into the RSV vector pBJ9 using XbaI and HindIII. The resulting plasmids were RSV E6 (p4483), RSV E7 (p4484) and RSV E7 DLYC (p4485). Coding sequences for the human p21CIP gene were subcloned from the pCDNAp21 expression vector, a gift from Dr P.Hinds, into the RSV vector using HindIII–XbaI digestion. The resulting plasmid is RSV p21 (p4486). Cell lines The human cervical cancer cell lines HeLa, Caski and C33A, as well as the human osteosarcoma cell line U2OS, were maintained as monolayers in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cellular growth suppression assay Cells were seeded at 1–2 105 per 60 mm plate the day before transfection. Transfections were performed using a total of 10 g of DNA and Fugene™ 6 (Boehringer Mannheim) according to the manufacturer's instructions. Transfection efficiencies of 70–90% were routinely achieved. Unless indicated otherwise, the cells were transfected with 5 g of E2 plasmid, 1 g of neomycin selection plasmid and 1 g of viral oncogene plasmid. Salmon sperm DNA was used as carrier DNA. At 24 h post-transfection, the cells were split into 3 10 cm plates and placed under selection in medium containing 900 g/ml G418 (Gemini Bio-Products, Inc.) for HeLa and U2OS cells, 400 g/ml for Caski and 1100 g/ml for C33A cells. Cells were washed once with phosphate-buffered saline (PBS) and overlayed with fresh selection medium every third day. The number of colonies could easily be determined after 2–3 weeks, after which time they were fixed using 10% formaldehyde in PBS, stained with methylene blue and counted. Senescence assay Staining for perinuclear SA-Gal activity was performed as previously described (Dimri et al., 1995). Immunoblotting and antibodies For western blot analysis, the cells were washed with PBS and scraped into RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 160 mM NaCl, 10 nM Tris pH 7.4, 5 mM EDTA) containing 16 g/ml benzamidine–HCl, 10 g/ml phenanthroline, 10 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin A and 1 mM phenylmethylsulfonyl fluoride. The lysates were incubated at 4°C for 30 min, cell debris was pelleted and the protein concentration of the supernatants was determined using Bradford assays (Bio-Rad). Protein lysates were normalized for equal protein concentrations and separated by SDS– PAGE. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and probed with the appropriate antibodies. Signals were detected by secondary horseradish peroxidase-conjugated antibody and enhanced chemiluminescence as recommended by the manufacturer (NEN™ Life Science Products, Inc.). The p21CIP monoclonal 6B6 antibody was from PharMingen. The monoclonal antibody to p16INK4A was a generous gift from Dr P.Hinds. Microinjection HeLa cells were plated in 35 mm glass bottom dishes (MatTek Corporation, MA) 24 h prior to injection. Cells were injected cytoplasmically with 110 ng/ml E2-TA plasmid alone or with 2 mg/ml morpholino oligonucleotides (GeneTools, LLC). The sequence of the antisense p21CIP oligonucleotide was 5'-CGCCTCCTCTGAGTGCCTCGGTGCC-3' and that of the standard control oligonucleotide was 5'-CCTCTTACCTCAGTTACAATTTATA-3'. FITC-conjugated dextran was used as a co-injection marker. Microinjections were performed using the CompiC Inject automated system (Luigs & Neumann) at the EMBL (Heidelberg, Germany). Following the injections, cells were cultured in 2% serum at 32°C for 5 days, and then switched to 37°C. Images were taken on day 7 post-injection using a Leica fluorescent microscope equipped with a 63 objective.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HeLa