The human prostate cell line PC-3, and the murine cell line RM-1, were obtained from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences, Shanghai, China. Fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM), and Roswell Park Memorial Institute medium 1640 (RPMI-1640) were bought from Gibco, USA; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide (EB), and acridine orange (AO), from Sigma, USA; and 4′-6-diamidino-2-phenylindole (DAPI) and Annexin V apoptosis assay, from BioVision, USA; six-week-old C57BL/6 mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing, China. The recombinant adenoviruses Ad-hTERTp-E1a-Apoptin, Ad-hTERTp-E1a-EGFP, Ad-CMV-Apoptin, and Ad-CMV-EGFP were constructed and saved in our laboratory (5) (Fig. 1).

We incubated 5×10⁵ PC-3 cells in RPMI-1640 and RM-1 cells in DMEM at 37°C in 5% CO₂; both culture media were supplemented with 10% fetal bovine serum to form complete media. Recombinant adenoviruses were diluted to 5×10⁷ pfu/ml with either DMEM or antibiotic. Infection was performed at a multiplicity of infection (MOI) of 100. The diluted recombinant adenoviruses were inoculated on the cell cultures and maintained at 37°C in 5% CO₂ for 4 h and then added to their respective complete medium, in which cultivation continued for 48 h.

PC-3 and RM-1 cells were seeded in 96-well plates (5×10³ cells/well) and infected with various concentrations (1, 10 and 100 MOI) of recombinant adeno-viruses. Viability was measured after 12, 24, 36, 48, 60, 72, 84 and 96 h by treating cells with 20 μl/well MTT (5 mg/ml) and incubating at 37°C in 5% CO₂ for 4 h. The culture media were removed, and the crystals formed were dissolved by adding 150 μl/well dimethylsulfoxide. Untreated PC-3 and RM-1 cells were used as controls and all measurements were performed in triplicate. The absorbance at 490 nm (A) was measured; untreated cells were used as controls. The percent cell survival was expressed using the following formula: (average absorbance value of control well - average absorbance value of the experimental well)/average absorbance value of control well (5).

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×10⁶ cells; MOI 100) were trypsinized, washed 2 times in phosphate-buffered saline (PBS), and the cell pellet obtained was resuspended in PBS. To the resuspended solution, we added 2 μl AO/EB solution (AO, 100 μg/ml; EB, 100 μg/ml; dissolved in PBS) and vortexed the resulting sample. Next, 20 μl of the sample was placed on a microscope slide with a cover slip, and images of representative cells were obtained with a digital video camera connected to the 100X objective lens of a fluorescence microscope. The images from the microscope were processed with the Image-Pro Plus (IPP, version 5.0.2) software program.

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×10⁶ cells; MOI, 100) were trypsinized, washed once in PBS, and the cell pellet obtained was resuspended in 200 μl binding buffer. To the resuspended solution, 2 μl fluorescein isothiocyanate (FITC)-labeled Annexin V and 2 μl propidium iodide (PI) were added. The resulting mixtures were incubated in the dark for 5 min at room temperature and examined under a laser scanning confocal microscope.

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×10⁶ cells; 100 MOI) were trypsinized and washed once in PBS as above. The cell pellet obtained was resuspended in 200 μl 25% glutaraldehyde, washed 3 times in PBS, resuspended again in 200 μl 100 ng/ml DAPI and a 20 μl aliquot of the resulting solution was placed on a microscope slide, coverslipped and imaged as described previously.

RM-1 cells were harvested by trypsinization and resuspended in serum-free DMEM after being washed with PBS. The cell concentration was adjusted to 5×10⁷ cells/ml. Within 1 h of harvesting, 100 μl of cell suspension was injected either subcutaneously into the right flank or into the caudal vein of C57BL/6 mice. When the resulting tumors reached a diameter of 2–5 mm (8 days), the mice were randomly divided into six groups of five mice each. Each mouse in five of these groups received treatment consisting of a single intratumoral and a caudal vein injection; the treatments were repeated two times a week for three weeks and then changed to once a week for three weeks. The five treatments used were 100 μl injections of: i) Ad-hTERT-E1a-Apoptin alone (10¹¹ pfu/mouse in saline), ii) Ad-CMV-Apoptin (10¹¹ pfu/mouse in saline), iii) Ad-hTERT-E1a-EGFP (10¹¹ pfu/mouse in saline), iv) Ad-CMV-EGFP (10¹¹ pfu/mouse in saline) and v) saline. The mice in the sixth group were untreated and served as controls. Tumor size was measured using calipers every 2 days. Tumor volumes were calculated as follows: [0.52 (smallest diameter of tumor)² (largest diameter of tumor)] (9). After 63 days, all mice were sacrificed and their cumulative survival was calculated.

The statistical significance of differences was determined using one-way analysis of variance (ANOVA), and statistical significance was accepted as P<0.05. Log-rank tests were used for survival analysis. Data from all animals are presented in Kaplan-Meier plots.

Cell viability was assessed using the MTT colorimetric assay. MTT is taken up into cells by endocytosis or by a protein-facilitated mechanism and reduced, mainly by mitochondrial enzymes, to yield a purple formazan product, which is largely impermeable to cell membranes and therefore accumulates within living cells. Solubilization of the cells liberates the purple product, which can be detected using a colorimetric measurement. The ability of cells to reduce MTT provides an indication of mitochondrial integrity and activity, which in turn may be interpreted as a measure of cell number/proliferation/viability/survival/toxicity (10).

As shown in Fig. 2, with longer infection times, the growth of PC-3 and RM-1 cells infected with Ad-CMV-Apoptin, Ad-hTERT-E1a-EGFP, Ad-CMV-EGFP and Ad-hTERT-E1a-Apoptin was inhibited. However, cells infected with replication-incompetent adenoviruses (Ad-CMV-Apoptin and Ad-CMV-EGFP) gradually resumed their growth after 48 h. In contrast, Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP were more effective in inhibiting cell growth. Cell viability depended on the MOI of the recombinant adeno-viruses to some extent. There was no significant difference in the growth of cells at different infection doses in the first 36 h (P>0.05). In contrast, after 48 h, the 100 MOI group showed significantly increased inhibition compared with the 1 MOI and 10 MOI groups. In the 100 MOI group, obvious suppression was seen after 24 h (P<0.05). With longer infection times, both Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP were more effective in inhibiting cell growth, but the former was more effective than the latter. In addition, Ad-CMV-Apoptin was more effective than Ad-CMV-EGFP. In PC-3 and RM-1 cells, infection with Ad-CMV-Apoptin at a MOI of 10 or 100 inhibited cell growth by 30-35% after 4 days. Infection with 1 MOI or 10 MOI of Ad-hTERT-E1a-EGFP and Ad-hTERT-E1a-Apoptin inhibited cell growth by 20–30 and 40–50% after 4 days, respectively and that with 100 MOI almost blocked cell growth (60–70%). Ad-CMV-EGFP, however, did not significantly inhibit cell growth. In conclusion, Ad-hTERT-E1a-Apoptin effectively restricts the growth of cultured PC-3 and RM-1 cells. The interaction between infection time and MOI was complex and synergistic and cell viability showed a non-rigorous dependent relationship with both factors. Therefore, we performed the following in vitro experiments 48 h after infection at 100 MOI.

For the analysis of cell death, we used fluorescent assays of AO/EB double staining. AO is taken up by both viable and non-viable cells, which emit green fluorescence if the dye is intercalated into double-stranded nucleic acid (DNA) and red fluorescence if it is bound to single-stranded nucleic acid (RNA). EB is taken up by only non-viable cells, which emit red fluorescence because of dye intercalation into DNA (11).

Chromatin condensation, nuclear fragmentation and membrane destruction are the hallmarks of apoptotic cells (8). Using AO/EB staining, we analyzed the effects of Ad-hTERTp-E1a-Apoptin and Ad-CMV-Apoptin infections on the nuclear and the membranes of PC-3 and RM-1 cells. As shown in Fig. 3A, normal cell membranes of PC-3 and RM-1 cells were intact and stained bright green with AO. Loss of cytoplasmic membrane integrity resulted in the uptake of EB by Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected PC-3 and RM-1 cells, with orange EB-stained cells dominating over bright green AO-stained cells. Using the AO/EB method, we also quantified the percentage of live, necrotic and apoptotic cells after Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin treatment (Fig. 3B). Infection with Ad-CMV-Apoptin was slightly cytotoxic and the main change in morphology indicated apoptosis more than necrosis (Fig. 3B, middle panels). In contrast, infection with Ad-hTERT-E1a-Apoptin was strongly cytotoxic and apoptosis occurred very quickly, so that the main change in morphology was necrosis rather than apoptosis (Fig. 3B, right panels).

This assay is based on the ability of the protein Annexin V to bind to phosphatidylserine (PS) exposed on the outer membrane leaflet of apoptotic cells (PS also appears on the necrotic cell surface). In viable cells, PS is located in the inner membrane leaflet, but upon induction of apoptosis, it is translocated to the outer membrane leaflet and becomes available for Annexin V binding. The addition of phosphatidylinositol (PI) enabled viable apoptotic cells to be distinguished from necrotic cells (12).

PC-3 and RM-1 cells infected with Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin were stained with Annexin V-FITC/PI and observed under a laser scanning confocal microscope. Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected cells displayed red fluorescence and fragmented chromatin when stained with PI (Fig. 4A, left panels) and green fluorescence when stained with Annexin V-FITC (Fig. 4A, middle panels). The green fluorescence was mainly concentrated in the cell membrane, a characteristic of phospholipid membranes valgus. PC-3 and RM-1 cells stained with Annexin V-FITC/PI showed a red nucleus (PI) and a halo of green (FITC) on the cell surface, which are indicative of phospholipid membranes valgus and fragmented chromatin. These results indicated that Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin induced apoptosis in PC-3 and RM-1 cells.

The blue fluorescent DAPI nucleic acid stain preferentially stains double-stranded DNA (dsDNA). The stain appears to associate with A/T clusters in the minor groove. Binding of DAPI to dsDNA produces ∼20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. DAPI also binds RNA but through a different mechanism, which is thought to involve A/U-selective intercalation. The DAPI/RNA complex exhibits a longer-wavelength fluorescence emission maximum than the DAPI/dsDNA complex (∼500 vs. ∼460 nm) and a quantum yield that is only ∼20% as high (13).

PC-3 and RM-1 cells infected with Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin were stained with DAPI and observed under a laser scanning confocal microscope. The nuclei of uninfected cells (controls) showed a uniform blue fluorescence and were structurally normal, while those of Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected cells displayed light blue fluorescence and condensed and fragmented chromatin (Fig. 4B). These results indicated that Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin induced apoptosis in PC-3 and RM-1 cells.

We next examined the antitumor potential of Ad-hTERT-E1a-Apoptin in the RM-1 tumor model. The growth kinetics of the tumors treated with intratumoral injections are shown in Fig. 5A. Compared with the saline control and Ad-CMV-EGFP groups, the recombinant adenovirus groups showed suppression of tumor growth; this effect was seen after the first three injections and continued up to the end of the treatment period. However, soon after the last injection, tumor growth gradually resumed in the recombinant virus groups, but was slowest in the Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP groups. The growth kinetics of the tumors treated with intravenous injections is shown in Fig. 5B. Compared with the tumors in the saline controls and Ad-CMV-EGFP-infected groups, those in the recombinant adenovirus groups were suppressed after the first three injections. Tumor suppression continued up to the end of the treatment period. However, soon after the last injection, the Ad-CMV-Apoptin- and Ad-CMV-EGFP-infected tumors gradually resumed their growth. Most of the Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-infected tumors also resumed growth, but these grew more slowly. The tumors in the intravenous injection groups grew more rapidly than those in the intratumoral injection groups. The main cause of this difference may be the faster effect of direct intratumoral injection than of intravenous injection. We also evaluated the ability of the recombinant adenoviruses to prolong the survival of the tumor-bearing mice (Fig. 5C). All saline-, Ad-CMV-Apoptin- and Ad-CMV-EGFP-treated animals died between 26 and 52 days after intratumoral injection. In contrast, 60 and 80% of Ad-hTERT-E1a-EGFP- and Ad-hTERT-E1a-Apoptin-infected animals, respectively, were still alive at that point (Fig. 5C). Mouse survival analysis showed that Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP treatments significantly increased mouse survival in the RM-1 tumor model in comparison with the other recombinant adenovirus treatments and saline treatment (Fig. 5D).

When the experiment was terminated on day 63, 80% of Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-infected animals were alive and the median survival time did not differ significantly between these two groups. None of the mice in the other groups were alive at the end of the experiment. In conclusion, inoculation with Ad-hTERT-E1a-Apoptin had significant survival benefits and reduced tumor size in vivo.

In the Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-treated groups, tumors lost tissue integrity and showed increased intercellular spaces containing remnants of disintegrating cells (Fig. 6A). None of these changes were seen in the Ad-CMV-EGFP-treated, Ad-CMV-Apoptin-treated, and saline control groups. These results indicated that Ad-hTERT-E1a-Apoptin had anti-tumor effects on solid tumors. This recombinant adenovirus also significantly decreased the tumor burden of the mice. Ad-hTERT-E1a-Apoptin-infected tumors showed minimal metastatic nodules, unlike the other treatment groups and the control group, which showed severe metastasis. Taken together, the systemic delivery of Ad-hTERT-E1a-Apoptin significantly reduced tumor burden and provided survival benefits in the RM-1 tumor model.