Figure 1.

hrR3 replication is greater in arrested p16-deleted cells than in arrested p16-expressing cells. (a) Recovery in plaque forming units (pfu) ml⁻¹ of infectious KOS-derived vRR(ICP6, UL39)^–LacZ⁺ hrR3 (from S Weller, University of Connecticut, Farmington, CT, USA) (Goldstein and Weller, 1988) 48 h after infecting (multiplicity of infection (MOI)=1.5) cultured human (U87 glioma, HF (human fibroblasts), T98 glioma, U373 glioma and U373-mRR cells; all obtained from American Type Culture Collection/ATCC, Manassas, VA, USA) and murine (MEFs (murine embryonic fibroblasts), obtained from R DePinho, Dan Farber Cancer Institute, Boston, MA, USA) cells that were either cycling or in G₀/G₁ (isolated by flow cytometry). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U ml⁻¹ of penicillin, and 100 μg ml⁻¹ of streptomycin at 37 °C and 5% CO₂. U373-mRR were generated by lipofectamine (Invitrogen, Carlsbad, CA, USA) transfection of U373 with the neomycin resistance gene-containing plasmid pCDNA3.1 (Invitrogen), with the cDNA for the M2 subunit of mRR subcloned into the plasmid. The M2 subunit of mRR cDNA was generated by RT–PCR of mRNAs obtained from U373 cells using a 5′ primer from positions 189 to 211 of the M2 subunit coding sequence including the AUG site (5′-ATCCGGATCCACTATGCTCTCCCTCCGTGT-3′) and the 3′ primer from positions 1346 to 1368 including the UAA site (5′-GCTTAAGCTTATTTAGAAGTCAGCATCCAAG-3′), with the resulting PCR product first cloned into the TA cloning vector (Invitrogen). MEFs were isolated as described (Sharpless et al., 2001). For FACS analysis, 10⁶ cells were trypsinized, centrifuged at 1000 g for 5 min, fixed by gradual addition of ice cold 70% ethanol (30 min at 4 °C) and washed with phosphate buffered saline (PBS). Cells were then treated with RNase (10 μg ml⁻¹) for 30 min at 37 °C, washed once with PBS, resuspended and stained in 1 ml of 69 μM propidium iodide in 38 mM sodium citrate for 30 min at room temperature. The cell cycle phase distribution was determined by analytical DNA flow cytometry as described (Gray-Bablin et al., 1997). Alternatively, to isolate G₀/G₁ cells by DNA content, 10⁹ cells were trypsinized, resuspended in PBS, and fluorescence activated cell sorting (FACS)-sorted. To measure viral yield, 10⁵ cells were plated into 12-well plates in DMEM plus 10% fetal calf serum. The next day, cells were infected with hrR3 (MOI=1.5) for 48 h. Infected cells were scraped into the medium and subjected to three freeze-thaw cycles. Virus titers were determined by plaque assays on Vero cells. Standard deviations are shown. (b) Ratio of viral yield in cycling cells to viral yield in G₀/G₁ cells for the six different cell lines analysed in (a). The p16+/+ expressing cells (HF, MEF, p53⁻ MEF, U373) exhibited significantly greater ratios than their p16−/− deleted counterparts (U87/T98 for HF, p16−/− MEF for MEF and for p53−/− MEF) or U373-mRR (P<0.05). Similar results (not shown) were obtained with murine fetal astrocytes of all p16/p53 phenotypes (obtained from R DePinho) and cells in G₁ obtained by treating with 40 μM lovastatin (Merck, Rathway, NJ, USA) for 36 h, with lovastatin converted to its active form as described previously (Gray-Bablin et al., 1997).