Figure 2
Prostatic acid phosphatase (PAP) expression. (a) Reverse transcriptase (RT)–PCR analysis of Vero cells infected with bPΔ6-mPAP shows mouse PAP (mPAP) mRNA expression. Cells were grown in six-well plates and infected at 90–95% confluence with either bPΔ6-hPAP, bPΔ6-mPAP or bPΔ6-empty viruses at a multiplicity of infection (MOI) of 2. After 2 h, virus inoculum was removed and replaced with medium Dulbecco's modified Eagle's medium (DMEM)/1% inactivated fetal calf serum (FCS). After 8 h, the total RNA fraction was isolated from cell lysates using TRIzol (Invitrogen, Carlsbad, CA, USA), and treated with RQ1 RNase-free DNase I (Promega, Madison, WI, USA). Reverse transcription was carried out using SuperScriptIII First Strand Synthesis kit (Invitrogen) and random hexamer primers. cDNAs were subjected to conventional PCR (5 min at 95 °C then 28 cycles: 30 s at 95 °C, 15 s at 68 °C, 20 s at 72 °C; and terminal elongation 2 min at 72 °C) with specific primers, P1mPAP619 5′-gcttcctggacaccttgtcgtcgctgtcg-3′ and P2 mPAP1214 5′-attccgtccttggtggctgc-3′, designed to generate a PCR product of 595 bp. As a positive control, plasmid DNA (pVec92-mPAP) was used and the PCR was performed without the reverse transcription step. The reaction product was analyzed by 1.2% agarose gel electrophoresis and stained with ethidium bromide. (b) Western blot analysis of hPAP expression in CT-26 colorectal carcinoma,13 RM-1 and TRAMP-C2 cell lines after infection with bPΔ6-hPAP (indicated by +) or bPΔ6-empty (indicated by −) at MOI=2. Human prostate adenocarcinoma LNCaP cells24 and human embryonic kidney 293 cells were used as controls. Cell lysates were prepared using RIPA buffer36 24 h after infection. Each lysate was separated by SDS–polyacrylamide electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), and immunoblotted with anti-hPAP (Dako, Carpinteria, CA, USA) Rabbit anti-human PAP, A0627) antibody (diluted 1:1000) using standard procedures. (c) Mouse prostate cancer cells express PAP. Total RNA was isolated from LNCaP, TRAMP-C2 and RM-1 cells and RT–PCR was performed using primers previously described.37 mPAP mRNA was observed in both TRAMP-C2 and RM-1 cells but not in human LNCaP prostate cancer cells. (d) In vivo expression of hPAP. TRAMP-C2 cells (5 × 106 per mouse) were implanted subcutaneously into the flanks of male 6- to 8-week-old C57/BL6 mice (National Cancer Institute, Frederick, MD, USA). Once tumors were established (50–10 mm3) mice were treated intra-neoplastically once with bPΔ6-hPAP (2 × 107 PFU). After 48 h, tumors were harvested, homogenized and centrifuged. Supernatants were assayed for hPAP using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to manufacturer's instructions. Error bars represent the standard deviation of three measurements. All in vivo procedures were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.
