Vpr has previously been demonstrated to induce G₂ cell cycle arrest as well as apoptosis in a number of tumor cells 2930. Previously, we found that C81, a carboxy-terminally truncated form of Vpr, is a strong inducer of apoptosis with G₁ arrest, but not G₂ arrest, of the cell cycle, and that the apoptotic activity of C81 is stronger than that of Vpr in human cells 35. Based on the growth arresting and apoptosis inducing properties in tumor cells, C81 and Vpr have potential therapeutic applications for treating cancer. To further investigate this potential, we examined whether Vpr and C81 can induce apoptosis in tumor cells. We transiently transfected two human tumor cell lines, HeLa and HepG2, as well as 293 T cells, with a pME18Neo expression vector encoding Flag-tagged wild-type Vpr, C81, or the empty vector pME18Neo-Flag as a control. We examined the expression of Vpr and C81, 48 h post-transfection by Western blot analysis using the anti-Flag M2 monoclonal antibody. Fig. 1 shows protein bands with molecular masses that are consistent with the calculated masses of the sequences of Flag-tagged Vpr and Flag-tagged C81.

Next, we detected apoptotic cells by monitoring fluorescence after staining the cells with Hoechst 33258. HeLa, HepG2, and 293 T cells were transfected with the pME18Neo empty vector, or the expression plasmids for Vpr or C81. At 48 h post-transfection, cells were fixed and stained with anti-Flag followed by Alexa-488-conjugated anti-mouse IgG. To monitor nuclear morphology, cells were stained with Hoechst 33258. The cells that expressed either Vpr or C81 had condensed chromatin, a hallmark of cells undergoing apoptosis (Fig. 2A). Furthermore, we assessed apoptosis in Vpr- and C81-expressing cells by measuring the activity of caspase-3, which plays an essential role in the induction of apoptosis (Fig. 2B). Caspase-3 activity was significantly higher in the Vpr- and C81-expressing cells compared to the control vector transfected cells. Caspase-3 activity was approximately two-five fold higher than in the cells transfected with the control pME18Neo empty vector. Interestingly, caspase-3 activity was significantly higher in cells that had been transfected with the C81 expression vector, compared to cells transfected with the Vpr expression vector. Conversely, treatment with the caspase-3 specific inhibitor, Z-DEVD-FMK, suppressed the activity of caspase-3 in all transfected cells, including cells transfected with the control pME18Neo empty vector. These results strongly suggested that Vpr and C81 can induce apoptosis in the HepG2 and HeLa cell lines.

The apoptotic activity of Vpr and C81 was further quantified with flow cytometry by combined staining of transfected samples with phycoerythrin-(PE)-Annexin V and 7-amino-actinomycin D (7-AAD) (Fig. 2C). A prominent event in early apoptosis is the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane. Cell surface exposed PS can be specifically detected with PE-Annexin V. At late stages of apoptosis, or in necrosis, cell membrane integrity is lost, allowing entry of the DNA-binding dye 7-AAD into cells. The population of Annexin V positive and 7-AAD negative apoptotic cells among cells expressing Vpr or C81 reached to approximately 5.52% and 7.15% in HeLa cells, respectively, and 10.32% and 12.45% in HepG2 cells, respectively. These values were much higher than in cells that had been transfected with the control pME18Neo empty vector or mock transfected (3.71% and 1.35% in HeLa cells, respectively, and 9.55% and 0% in HepG2 cells, respectively). Interestingly, the populations of apoptotic cells in both tumor cell lines were significantly higher in cells transfected with the C81 expression vector, compared to cells transfected with the Vpr expression vector. Similar results were obtained with 293 T cells. Only a small population of 293 T cells transfected with either Vpr, C81, or the control vector bound Annexin V (2.02%, 2.28%, and 1.65%, respectively). Since these results show the same trends as the Hoechst 33258 staining (Fig. 2A) and the caspase-3 activity (Fig. 2B), we conclude that Vpr and C81 induce apoptosis in the human tumor cell lines HepG2 and HeLa.

Vpr and C81 are reported to target mitochondria to induce apoptosis 1034. To examine whether Vpr and C81 target mitochondria in tumor cells, we measured caspase-8 and caspase-9 activity. Caspase-8 is activated by FasL and tumor necrosis factor-α (TNF-α), whereas caspase-9 is activated by the disruption of mitochondrial function. In tumor cells, as well as 293 T cells, caspase-9 activity induced by Vpr or C81 was higher than caspase-8 activity (Fig. 3). Caspase-9 activity in Vpr or C81-transfected cells was significantly higher than in the control vector transfected cells (p < 0.01). In addition, although apoptosis induced by Vpr or C81 was significantly inhibited when inhibitors of caspase-9 were added to the Vpr- or C81-transfected cells (p < 0.01), a caspase-8 inhibitor only slightly inhibited apoptosis in these transfected cells. These results suggest that both Vpr and C81 induce apoptosis via the activation of caspase-9.

Vpr induces G₂ cell cycle arrest in various human cells 10, whereas C81 induces G₁ cell cycle arrest in human and rodent cells 3435. To determine whether Vpr and C81 induce cell cycle arrest in HeLa, HepG2, or 293 T cells, we transfected these cells with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control plasmid pME18Neo-Flag, together with a small amount of a green fluorescent protein (GFP) expression vector, pEGFP-N1, to identify transfected cells. Cells were fixed and stained with propidium iodide (PI) 48 h post-transfection and analyzed by flow cytometry. We measured the DNA content of GFP-positive cells to determine the distribution of cells across the cell cycle (Fig. 4A). C81 failed to arrest the cell cycle at the G₂ phase in HeLa, 293 T, or HepG2 cells. To confirm this result, we prepared total cell extracts and examined cyclin B1, which controls the cell cycle in the G₂ phase, by Western blot analysis (Fig. 4B). C81 failed to induce accumulation of cyclin B1 in HeLa, 293 T, or HepG2 cells, suggesting that C81 failed to arrest the cell cycle at the G₂ phase in these cells. In contrast, transfection with Vpr led to an increase in the number of HeLa and 293 T cells in the G₂ phase of the cell cycle, compared with cells that had been transfected with the control pME18Neo-Flag. Moreover, Vpr induced accumulation of cyclin B1 in HeLa and 293 T cells. However, Vpr was unable to arrest the cell cycle at the G₂ phase, or to induce accumulation of cyclin B1, in HepG2 cells (Fig. 4).

It has recently been reported that Vpr targets the DCAF1 adaptor of the Cul4A-DDB1 ligase to induce cell cycle arrest at the G₂ phase 28. Thus, we examined the expression levels of DDB1 in various cell lines, including HepG2 cells, which do not arrest at G₂ when transfected with Vpr (Fig. 5A). Interestingly, Western blot analysis revealed that Vpr, but not C81, could inhibit DDB1 expression in HepG2 cells. In contrast to Vpr, C81 had no effect on the expression of DDB1 in HepG2 cells.

To determine the effect of DDB1 in HepG2 cells, we constructed a DDB1 expression vector, pDDB1-IRES-GFP, which was co-transfected with either the Vpr or C81 expression vector. As shown in Fig. 5B, overexpression of DDB1 resulted in cell cycle arrest at G₂ in HepG2 cells transfected with Vpr. Overexpression of DDB1 had no effect on the induction of G₂ arrest by Vpr in HeLa or 293 T cells. This result clearly suggests that DDB1 plays an essential role in Vpr-induced cell cycle arrest. Strikingly, C81, which was completely inactive for G₂ arrest in all cell lines tested, was able to induce G₂ arrest in HepG2 cells overexpressing DDB1, but not in HeLa or 293 T cells overexpressing DDB1. These results suggest that C81 can cause G₂ arrest independent of its ability to induce apoptosis via G₁ arrest of the cell cycle, although the former activity is cell type dependent.

The expression vector pME18Neo encoding a Flag-tagged wild-type Vpr or C81, and the control vector pME18Neo have been described previously 4041. A red-shifted variant of wild-type GFP that has been modified for brighter fluorescence was encoded by pEGFP-N1 and used as a reporter to identify transfected cells 42. Bacterial β-galactosidase encoded by pSV-β-galactosidase was included for normalization of the transfection efficiency 40. The mRNA of human DDB1 was amplified by RT-PCR from RNA derived from Jurkat cells. RT was performed with an oligo-dT primer and PCR was performed using the following primers: 5'-gcggccgcacatgtcgtacaactacg-3' and 5'-gtcgacgctaatggatccgagttagc. The resulting amplicon served as the template for a second PCR reaction using the primers 5'-ataagaatgcggccgcacatgtcgt-3' and 5'-tggccgacgtcgacgctaatggatc-3'. The second PCR product was subcloned between the NotI-SalI sites of pIRES-hrGFP-2a (Stratagene, La Jolla, CA), generating pDDB1-IRES-GFP.

HeLa cells, which are human epithelial cervical carcinoma cells 4344, and 293 T cells that stably express the large T-antigen of simian sarcoma virus 40 45 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 μg/ml). HepG2 cells, which were derived from a well differentiated hepatocellular carcinoma 4647 were obtained from the RIKEN Bio Resource Center (Ibaraki, Japan) and maintained in DMEM supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), and 0.1 mM non-essential amino acids. Transfection of cells was performed using FuGENE HD (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions.

At 24 or 48 h post-transfection with the indicated plasmids and pSV-β-galactosidase, cells were washed with phosphate-buffered saline (PBS) and divided into two aliquots. One aliquot was subjected to an assay for β-galactosidase activity with the FluoReporter LacZ/Galactosidase Quantitation kit (Molecular Probes, Eugene, OR), to monitor the transfection efficiency. The other aliquot of cells was lysed for 10 min on ice in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% desoxycholate supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitor cocktail1 (Sigma, St. Louis, Mo). Lysates were centrifuged for 10 min at 20,000 g, mixed with sample buffer for SDS-PAGE and then boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL), with bovine serum albumin (BSA) as the standard. Aliquots with equal β-galactosidase activity were examined by Western blotting as described previously 3539. An anti-Flag MAb (M2) (Sigma, St. Louis, MO), anti-Cyclin-B1 (H-20) antibody (Santa Cruz biotechnology, Santa Cruz, CA), anti-DDB1 MAb (ZYMED Laboratories Invitrogen, Carlsbad, CA), horseradish-peroxidase (HRP)-conjugated sheep anti-mouse IgG (Amersham Bioscience, Uppsala, Sweden) and HRP-conjugated goat anti-rabbit IgGs (Cell Signaling Technology, Danvers, MA) were used for Western blotting. Signals were visualized after treatment of the membrane with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).

The activities of caspase-3, 8, and 9 were determined using caspase-3/CPP32, caspase-8/Flice or caspase-9/Mch6 fluorometric assay kits (Bio Vision, Mountain View, CA) according to the manufacturer's protocols. Briefly, 30 h or 40 h post-transfection, in the presence or absence of 2 μM caspase-3, 8, or 9 specific inhibitors (Bio Vision, Mountain View, CA), cells were harvested and lysed with cell lysis buffer. Each cell lysate was incubated with the substrate for 2 h at 37°C. Measurements were made using a multilabel cell counter (Model1420; Wallac Arvo; Parkin Elmer, Wellesley, MA).

Hoechst 33258 was used to visualize the condensation and clumping of chromatin. At 48 h post-transfection with pME18Neo encoding Flag-tagged wild-type Vpr, C81, or the control pME18Neo-Flag, HeLa cells that were grown on coverslips were fixed with 4% formaldehyde followed by 70% ethanol. Flag-tagged-Vpr or C81 was detected by indirect immunofluorescent staining with the Flag-specific MAb M2 (Sigma, St. Louis, MO), followed by Alexa-488-conjugated anti-mouse IgG. The cells were then stained with Hoechst 33258 (Sigma, St. Louis, MO). Apoptotic bodies were characterized using confocal laser scanning microscopy (FV 1000; Olympus, Tokyo, Japan).

To detect Annexin V positive apoptotic cells, we used flow cytometry. At the indicated times post-transfection, cell monolayers were washed with PBS, detached with trypsin and EDTA, and 1 × 10⁵ cells were stained with 7-amino-actinomycin D (7-AAD) and Annexin V-PE (Nippon BD company Ltd, Japan) according to the manufacturer's instructions. Incubation of cell monolayers for 8 to 13 hours with actinomycin D (2 μg/ml) provided positive controls for apoptosis. For each sample, 10,000 GFP positive cells were analyzed using a FACS Calibur instrument (BD Biosciences, Japan) and the FCS Express version 3 software (De Novo software, CA).

At 48 h post-transfection, cells were harvested and fixed with 1% formaldehyde and then 70% ethanol. Fixed cells were incubated in PBS that contained RNase A (50 μg/ml) at 37°C for 30 min and then stained with PI (50 μg/ml). The fluorescence of 20,000 cells was analyzed using a FACS Calibur instrument (Becton-Dickinson, Mountain View, CA) with the CELL Quest software (Becton-Dickinson, Mountain View, CA). Data were extracted after gating to eliminate cells in which GFP emitted strong fluorescence. Ratios of the numbers of cells in the G₁ and G₂/M phase (G₂/M:G₁ ratios) were calculated using the ModFit LT Software (Verity Software House, Topsham, ME).

Statistical analyses were conducted using R version 2.8(1) 48.