Vpr induces cell cycle G2 arrest in various cancer cell types.

<p>All cancer cell lines (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-t001" target="_blank">Table 1</a> for details; only 3 cell lines are shown here as examples) were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#s4" target="_blank">Materials and Methods</a>. Cells were transduced with Adv or Adv-Vpr with MOI of 1.0. The cells were harvested 48 hrs post-infection (<i>p.i.</i>), Cells were then prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#s4" target="_blank">Materials and Methods</a>. Cellular DNA content was analyzed by FACScan flow cytometry (Becton Dickinson) as we previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Li1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Liang1" target="_blank">[19]</a>. The cell cycle profiles were modeled using ModFit software (Verity Software House, Inc.).</p

Vpr suppresses tumor regression in a neuroblastoma mouse model.

<p>Suppression of neuroblastoma tumor growth by Vpr is demonstrated here either by pre-transduction of Adv-Vpr (<b>A-B</b>) or post-intratumoral injection (<b>C</b>). For pre-transduction, wild type (WT) or DOX-resistant SK-N-SH were grown in DMEM supplemented with 10% FBS at 37°C with a 95%Air/5%CO₂ atmosphere. Fresh cells were first grown in a 12-well plate for 36 hours and adenoviral transduction was carried out 5 hours before cell inoculation with MOI of 2.5, which was determined empirically. Cells were then treated with Trpsin- EDTA, re-suspended in DMEM and washed with PBS 3 times. Final cells were suspended in DMEM for inoculation. About 2×10⁶ cells in the volume of 100 µl were injected <i>s.c.</i> in the left flank of C57-SCID mice. 3–4 mice were injected for each treatment. These treatment groups include an Adv viral control, Adv-Vpr and Adv-F34IVpr. The F34IVpr mutant was used here as a control since a single amino acid change of amino acid 34 from Phenylalanine (F) to Isoleucine (I) renders Vpr unable to cause apoptosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-g003" target="_blank">Figure 3C</a>) but allows for the cell cycle to enter a prolonged G2 phase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Benko1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Chen1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Vodicka1" target="_blank">[28]</a>. The tumor size was measured every 7 days by measuring two perpendicular tumor diameters using calipers. Final tumor measurement was at 26 days post-transduction and mice were then sacrificed for further analysis. For intra-lesional injection of Vpr, the WT and DOX-R SK-N-SH neuroblastoma cells were prepared essentially the same way as described above. 200 µl of the Adv, Adv-Vpr or Adv-F34Ivpr was then injected discretely 3-times into the tumors 2 weeks after cell inoculation with a viral concentration of 1,012/ml. The tumor size was measured every 7 days. Final measurement of tumor size was at 39 days post-injection and mice were then sacrificed for further analysis. Three independent experiments were carried out and results of these experiments with average tumor size with standard deviation (SD) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-t002" target="_blank">Table 2</a>.</p

Summary of Vpr-induced tumor regression of WT and DOX-resistant neuroblastoma in C57-SCID mice.

<p><b>Note:</b> Tumor sizes were measured at 39 days post-intratumoral injection (Week 0). Levels of statistical significance of the t-test results between the Adv control and the testing groups (Ad-Vpr or Ad-F34IVpr): *, p<0.05; **, p<0.001; Weighted average sums of the t-tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Tan1" target="_blank">[31]</a> for both wild type and Dox-R mice showed statistic differences at the level of p<0.05. Note: na, non-applicable.</p

Quantitative summary of Vpr-induced G2 arrest in various cancer types.

<p><b>Note:</b> *, PP2A is mutated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Ostrakhovitch1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Suzuki1" target="_blank">[34]</a>; **, F34IVpr causes cell cycle G2 shift but no cycling arrest <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Benko1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Chen1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Vodicka1" target="_blank">[28]</a>; WT, wild type; DOX-R, doxorubicin-resistant; G2 arrest: (+), strong G2 arrest; (−), no G2 arrest, (+/−), attenuated G2.</p

Vpr induces cell death and apoptosis in DOX-naïve and resistant SK-N-SH cells.

<p><b>A.</b> Vpr induces dose-dependent cell death in drug-naïve and resistant SK-N-SH cells. <b>a.</b> Level of Vpr-induced cell death was examined by infecting DOX-naïve and resistant SK-N-SH cells using increasing MOI of Adv or Adv-Vpr viruses. Cell death was measured by determining the cell membrane integrity and proliferation using Trypan blue straining (left) or cell viability by the MTT assay (right). Cells proliferation and viability were examined 5 days <i>p.i</i>. <b>b.</b> Vpr protein production was confirmed by Western blot analysis. Note that it is very difficult to detect Vpr protein at low MOI. Successful infection of Adv-Vpr was verified by enlarged nuclei of cells as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Bartz1" target="_blank">[43]</a>. <b>B.</b> Vpr induces cell death over time in drug- naïve and resistant SK-N-SH cells. Both drug-naïve and resistant SK-N-SH cells treated the same way as <b>A</b>. Only MOI2.5 was used here. <b>C.</b> Vpr induces apoptosis in drug-naïve and resistant SK-N-SH cells. Caspase-3 cleavage was monitored up to 36 hours. Initials: Csp3, caspase-3; cl-Csp3, cleaved caspase-3; m, Adv-VprF34I; w, Adv-Vpr.</p

Expression of Vpr leads to cell death in a variety of wide type (WT) and doxorubicin (DOX)-resistant cancer cells as indicated.

<p>Three pairs of drug naïve (WT) and DOX-resistant (DOX-R) cancer cells were tested for Vpr-induced G2 arrest (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-t001" target="_blank">Table 1</a>) and cell death. These cancer cell lines were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#s4" target="_blank">Materials and Methods</a>. Cells were transduced with Adv or Adv-Vpr with MOI of 2.5. Cell viability was determined either by the Trypan Blue exclusion assay, which identifies dead cells (top figure) or by the MTT assay to measure cell survival (bottom figure). Cells were examined 5 days <i>p.i.</i> Intials: WT, wildtype; Dox-R, doxorubicin-resistant; 1, Mock; 2, Adv and 3, Adv-Vpr.</p