Normal human prostate epithelial cells (PrEC) and hepatocytes (HC) were purchased from Lonza (Basel, Switzerland) and cultivated using the medium recommended by the supplier. Human cancer cell lines (LNCaP, PC3, HEP3B, HEPG2) were provided by the American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) and RPMI-1640 medium (Nissui, Tokyo, Japan) were used as the cultures for the cancer cell lines. The cell lines were grown in media supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaille, France), as previously described (5).

The full-length cDNA of the human REIC/Dkk-3 gene was inserted into the cosmid vector, pAxCAwt, and then transferred into an adenoviral vector using the COS-TPC method (Takara Bio, Shiga, Japan) (3). An adenoviral vector carrying the LacZ gene (Ad-LacZ) was used as the control vector. The adenoviral vectors consisted of replication-defective adenovirus of serotype 5 that contained the indicated genes under the control of the CMV early enhancer/chicken β actin (CAG) promoter.

The total protein of in vitro treated cells and mouse prostatic tissue was extracted with a lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 mM Na₃VO₄, 1 mM NaF, 10 mM β-GP). After centrifugation was completed, the volume of the supernatants was adjusted in each sample to achieve equal protein concentrations, and the samples were diluted with the same volume of 2X SDS sample buffer and heated for 5 min at 95˚C. The samples (10 μg of protein) were then separated on 7.5% SDS-PAGE gels and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The blots were blocked for 1 h with 10% non-fat milk powder, 6% gycine, and 0.1% Tween-20 in Tris-buffered saline (TBS) at room temperature. The REIC protein was then identified with the use of a primary antibody: rabbit anti-human REIC/Dkk-3 polyclonal antibody raised in our laboratory (3,5). After being extensively washed with 0.1% Tween-20 in TBS (T-TBS), the blots were exposed to horseradish peroxidase-conjugated secondary antibodies. After being extensively washed with T-TBS, the membranes were developed using the enhanced chemiluminescence detection method (ECL kit, Amersham Pharmacia Biotech, Chandler, AZ).

To examine the induction of in vitro apoptosis following the treatments, the cells were seeded in flat-bottom 6-well plates and incubated for 24 h. The cells were then treated with either the Ad-CAG-LacZ or Ad-CAG-REIC at the indicated multiplicity of infection (MOI) in the complete medium (500 μl) for 1 h. Fresh medium was added to bring the total to 2 ml. After an additional 72 h of incubation, Hoechst 33342 stock solution was added to the medium at a concentration of 2 μg/ml and the cells were incubated in the dark for 10 min. Hoechst 33342 is an intercalating dye that allows a determination of the variation in the total chromatin quantity and the degree of chromatin condensation to be made (17,18). Using fluorescence microscopy, apoptotic cells were identified by the presence of highly condensed or fragmented nuclei. Apoptotic cells were counted in five different fields under microscopic observation, and 100 cells were judged in each field.

An analysis of the biodistribution of Ad-REIC and a histological evaluation were completed following two doses of vector injection into the dorsolateral prostate (left lobe) and one dose of intravenous injection into the tail vein of each mouse. The treatment groups were classified as follows: control group (group 1), intraprostatic injection of the vehicle [phosphate buffered saline (PBS)]; intraprostatic injection of the vector (group 2), intraprostatic injection of 2.5×10⁸ viral particles (vp) of Ad-REIC in 20 μl vehicles; intraprostatic injection (group 3), intraprostatic injection of 2.0×10¹⁰ vp of Ad-REIC in 20 μl vehicles; intravenous injection via the tail vein (group 4): intravenous injection of 2.5×10⁸ vp of Ad-REIC in 100 μl vehicles. A total of 27 mice (C57BL/6 male mice, 6–8 weeks of age) were treated in order to obtain 19 evaluable animals, one for the control and two for each of the Ad-REIC treatment groups for each time-point. The first day of vector administration was designated as day 0, and the mice were sacrificed on days 1, 7 and 28. The physical appearance, activity levels, and mortality patterns of all of the animals were observed daily. When the mice were sacrificed, the following organs were collected for biodistribution and histological analysis: brain, lung, thymus, heart, liver, pancreas, spleen, kidney, stomach, colon, urinary bladder, dorsolateral prostate (injection site in groups 1–3), testis, femoris muscle, adrenal gland, and whole blood. To complete the DNA-PCR assays of the biodistribution analyses, whole blood was firstly drawn from the inferior vena cava of each animal under deep anesthesia with sodium pentobarbital. The blood samples were collected in a plastic tube and preserved with ethylenediaminetetra-acetic acid (EDTA). The organs were then dissected and stored at −80˚C. Scalpels and forceps were changed after each organ dissection or incision to avoid cross-contamination of the tissues. To complete the histological analysis of the potential side effects of Ad-REIC treatment, the tissues were fixed in formalin and embedded to create paraffin sections. The sections (5 μm) were stained with hematoxylin and eosin and examined for histopathological and cytotoxic changes.

DNA-PCR was performed on each mouse organ to detect the presence of vector DNA derived from the Ad-REIC vectors and to determine the biodistribution of Ad-REIC. The organs were weighed and each (~25 mg) was suspended in 180 μl of ATL buffer (QIAamp DNA Mini kit, Qiagen) with 20 μl of proteinase K. A 200-μl aliquot of each organ was incubated at 56˚C until the tissue was completely lysed and then was placed at room temperature. AL buffer (200 μl) was added to each sample and the samples were incubated at 70˚C for 10 min. 100% ethanol (200 μl) was added to each sample and the samples were mixed thoroughly and then placed into QIAamp spin-columns. After several washing steps were completed, the DNA was eluted with 200 μl of AE buffer. Each DNA sample was used in a PCR that amplified a 621-bp fragment of the REIC gene. The reactions contained 300 ng of sample DNA, 10 pmol of each primer (REIC-F: 5′-ATGCAGCGGCTTGGGGCCACCCTGCT GTGC-3′; REIC-R: 5′-GATGGTCCCATTGCTGCCCCTGGT GGCCAT-3′), 8 mmol of each deoxynucleoside triphosphate, 2X GC buffer, and 1 U of LA Taq (Takara Bio) in a volume of 20 μl. The reactions were incubated at 98˚C for 30 sec, then 30 cycles of 10 sec at 98˚C and 30 sec at 72˚C were performed. Following the final cycle, a 5-min incubation at 72˚C was performed. After loading dye was added to the reactions, the samples were electrophoresed in a 1.0% agarose gel containing 0.5 μg/ml ethidium bromide and visualized with an ultraviolet transilluminator. A sample containing an Ad-REIC vector was assayed as a positive control. A sample without any vector was assayed as a negative control in order to detect any contamination.

The data are expressed as the means ± the standard deviations (SD). An unpaired Student’s t-test was used to compare the data between the two groups. Differences were considered to be statistically significant at p<0.05.

To confirm whether protein expression and apoptotic effects were induced by Ad-REIC, various human cancer cell lines and normal human cells were treated with Ad-REIC. Western blot analysis demonstrated a significant expression of REIC protein in PC3 human prostate cancer cells and human hepatocytes on day 1 (Fig. 1A). In an apoptosis assay using the Hoechst 33342 stain, significant apoptosis induction was observed in Ad-REIC treated prostate cancer cells (LNCaP, PC3) and hepatoma cells (HEP3B, HEPG2) in comparison to the control Ad-LacZ treated cells; however, no apoptosis was observed in normal cells (PrEC, HC) (Fig. 1B). The mean rate of apoptosis at 100 MOI in the cancer cells (LNCaP, PC3, HEP3B, HEPG2) was 31.0, 67.0, 30.0 and 31.8%, respectively.

Human REIC gene detection was performed using DNA-PCR to identify the presence of Ad-REIC vectors in various tissues, including the brain, lung, thymus, heart, liver, pancreas, spleen, kidney, stomach, colon, urinary bladder, prostate (injection site in groups 1–3), testis, femoris muscle, adrenal gland, and whole blood. Total DNA was extracted from each tissue and analyzed for the presence of REIC DNA (621-bp fragment) using PCR. The PCR bands were confirmed with electrophoresis. The analysis used to detect REIC DNA in a given tissue is semi-quantitative and the assay provides a gross indication of the relative distribution of the vector Ad-REIC DNA. The sensitivity of the DNA-PCR was established by adding various amounts of Ad-REIC viral particles into the buffer (ranging from 1×10¹⁰ vp/ml to 1×10⁴ vp/ml. The limit of detection for this method was demonstrated to be 1×10⁷ vp/ml; however, trace levels were also visible at 1×10⁶ vp/ml (data not shown).

In the control group (group 1), no bands of vector DNA derived from Ad-REIC were detected, demonstrating that the assay did not react with the mouse REIC gene and no interference from human REIC DNA occurred in the mice. On day 1 after a dose of 2.5×10⁸ vp was injected into the prostate of the mice (group 2), vector Ad-REIC DNA was detected in the prostate (injection site), with trace amounts found in the brain, kidney, colon, and urinary bladder. Bands of vector DNA were also detected in the prostate on days 7 and 28. On day 1, the group that received the higher dose of 2.0×10¹⁰ vp of Ad-REIC (group 3) showed detectable levels of vector DNA in the lung, heart, liver, pancreas, spleen, kidney, stomach, colon, urinary bladder, prostate (injection site), testis, femoris muscle, and adrenal gland. On days 1, 7 and 28, the most pronounced levels of vector DNA were observed in the colon, urinary bladder, and prostate. On day 28, vector Ad-REIC DNA was still detectable in the colon, urinary bladder, and prostate.

The intravenous injection of Ad-REIC at a dose of 2.5×10⁸ vp (group 4) resulted in detectable levels of vector Ad-REIC DNA in the liver, spleen, colon, and whole blood on day 1. No detectable DNA vectors were present by day 7. A summary of the biodistribution of the Ad-REIC vectors following intraprostatic and intravenous administration is shown in Table I.

As a preliminary experiment, the expression of REIC protein following intraprostatic Ad-REIC injection was examined in normal mouse prostate using western blot analysis. On day 3, an elevated expression of intraprostatic REIC protein, in comparison to the control treatment, was verified (Fig. 5A). In all of the experiments, the physical appearance, activity, and mortality of the mice (groups 1–4) were evaluated daily. No adverse findings or signs of toxicity were noted and no animal deaths occurred in this study. At necropsy, no inflammation-related findings, such as the presence of abscesses or abundant ascites, were detected in any of the animals.

To examine the toxicology of the Ad-REIC treatment, histological analyses were completed on sections of the brain, lung, thymus, heart, liver, pancreas, spleen, kidney, stomach, colon, urinary bladder, dorsolateral prostate (injection site in groups 1–3), testis, femoris muscle, adrenal gland, and whole blood. We first analyzed the local effects of Ad-REIC injection using a histopathological analysis of the dorsolateral prostate, the vector injection site. In the Ad-REIC injected prostate of group 3, infiltrations of inflammatory cells were observed in the interstitial areas on day 1; however, no such infiltrations were observed on day 7 (Fig. 5B). Similar findings were also observed in the Ad-LacZ injected prostate; however, we were unable to detect any histological effects in the prostatic sections of the PBS treated group (group 1) (data not shown). Since the biodistribution of the Ad-REIC vectors following intraprostatic injection with the high dose (group 3) was observed to be widespread, we next investigated whether systemic toxicity resulted from Ad-REIC treatment in this group. As for tissues other than the prostate, no histopathological abnormalities were observed in the sections obtained from any treatment group at any time-point (Fig. 5C).

The liver is the primary target organ for adenoviral vectors following intravenous administration (19). We therefore examined the liver tissue of group 4. None of these mice showed any histopathological liver abnormalities, such as cytotoxicity or inflammation, during the experiment (Fig. 5C).