Two mouse strains were used in this study. Wild type (WT) C57BL/6 J mice were initially obtained from Dr. Sulev Kuuse, Institute of Molecular and Cell Biology, University of Tartu, (Estonia). RGS16 knockout (KO) mice generated on C57BL/6 genetic background were obtained from Pr. Kirk Druey, NIAID, Bethesda (USA). All mice used in this study were grown in the fish facilities if Tallinn Technical university. Before experiments RGS16 KO mice were backcrossed 6 generation to our C57Bl6/J from Tartu University, to ensure an homogeneous background in WT and RGS16KO. The total number of the mice used in this study was 44 (20 for the comparison of tumor growth in WT and RGS16 KO mice, and 24 for the comparison of tumor growth in WT and RGS16−/− mice treated with PCV2ORF3 or control plasmid).
Animal handling and maintenance were performed according to the interdisciplinary principles and guidelines for the use of animals in research, testing and education (FELASA) prepared by the Ad Hoc Committee on Animal Research (The New York Academy of Sciences, New York, NY, USA). The animal experiments described in this study were authorized by the Ethical and Animal Welfare Committee of Estonia (University of Tartu, ERC PERMIT nr. 181 T-1). Mice were euthanized by cervical dislocation, according to our approved protocols.
Cultivation of cell lines
B16F10 mouse melanoma cells and A375M human metastatic melanoma cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium; PAA). THP-1 human acute monocytic leukemia cells were cultured in RPMI 1640 (Roswell Park Memorial Institute medium 1640; PAA). All media were supplemented with 12% fetal calf serum (FCS; Gibco) and 1% penicillin-streptomycin (Sigma). M2 human filamin-A-deficient melanoma cell line was cultured in MEM (Minimal Essential Medium; PAA), supplemented with 8% newborn calf serum (NBCS; Gibco), 2% FCS and 1% penicillin-streptomycin (Sigma). After transfection, cells were cultured in 1/1 mix of new medium and conditioned medium (culture supernatant collected from cell plates), greatly enhancing cell survival. The cells were maintained at 37 °C in a humidified incubator of 5% CO2 in 95% air and subcultured twice per week. B16F10, A375, and M2 cells were provided by Dr. Teet Velling (Department of Gene Technology, Tallinn University of Technology, Estonia), THP-1 cells were provided by Prof Jean-Marc Cavaillon (Institute Pasteur, Paris, France).
Generation of the stable B16F10-luc cell line
B16F10 cell line expressing stably firefly luciferase (B16F10-luc) was generated using HIV1-based self-inactivating lentiviral vector system as described in . Sequence encoding luciferase from the plasmid pGL3 (Promega) was cloned under the control of the strong constitutive human elongation factor 1-α promoter in a transfer vector. The virus was produced by transient transfection of the transfer vector with accessory plasmids in HEK293T cells and pseudotyped with VSV-G envelope protein. Viral supernatant was used to infect B16F10 cells from which single cell-derived positive clone was isolated.
Isolation and cultivation of primary cells
RGS16 knockout (KO) and wild-type (WT) C57BL/6 mice were anesthetized using carbon dioxide and euthanized by cervical dislocation. Spleens from mice were removed and cell suspensions were obtained by pressing the spleens through a cell strainer in DMEM (PAA). Erythrocytes were lysed with ACK (Ammonium-Chloride-Potassium) lysis buffer (Lonza) for 10 min at room temperature. Then, cells were pelleted by centrifugation, washed twice with PBS and cultured in DMEM supplemented with 10% FCS (Gibco) at 37 °C in a humidified incubator of 5% CO2 in 95% air.
Construction of expression plasmids
To generate the pcDNA-ORF3 expression construct, the ORF3 of PCV2 was PCR amplified from the purified PCV2 genome (GenBank, AF055392). The PCR products were cloned into mammalian expression vector pcDNA3.1 (Invitrogen) using the KpnI/XhoI sites. Additionally, ORF3 was also cloned into the KpnI/XhoI sites of pcDNA3.1-His plasmid (Invitrogen) and the coding region of mCherry, excised from the pBSK-mCherry E3 vector, was added using the XhoI/XbaI sites, thus giving rise the expression plasmid pcDNA3-his-ORF3-mCherry. To obtain the pCEP-GFP-RGS16 expression construct, the full-length cDNA of porcine RGS16 (GenBank accession EU271873) was RT-PCR-amplified from LPS-activated poPBMCs. The PCR products were cloned into the XhoI/EcoRI sites of the pCEP-GFP plasmid (BD Biosciences). All of the used PCR primers were designed on the basis of ORF3- and RGS16-encoding sequences and the appropriate plasmid sequences.
Transfection of cells
The plasmid pcDNA-ORF3 was transiently transfected into B16F10, A375M and M2 melanoma cells using Lipofectamine ™ LTX (Invitrogen) in accordance with the manufacturer’s instructions. For comparison, pcDNA-ORF3 was also transiently transfected into THP-1 human acute monocytic leukemia cells. As a negative control, parallel transfections with empty pcDNA3.1 plasmid were carried out. The cells were seeded in six-well plates at a density of 5 × 105 cells per well. For each well of cells, 2.5 μg of plasmid DNA was diluted in 500 μl Opti-MEM®I Reduced Serum Medium, 2.5 μl of PLUS™ Reagent was added, and the mixture was incubated at room temperature for 10 min. 10 μl of Lipofectamine™ LTX was then added into the DNA solution and DNA: Lipofectamine complex was allowed to form by incubating at room temperature for 30 min. 500 μl of the DNA-Lipofectamine™ LTX complex was added to each well and the cells were then maintained at 37 °C in a humidified incubator with 5% CO2 in 95% air. After 24–48 h of transfection, cells were harvested, transfection efficiency was determined (Additional file 1: Figure S1) and cells were used for various assays described below.
Investigation of apoptosis by AnnexinV/PI staining flow cytometry assay
The extent of apoptosis was determined by flow cytometry using FITC-labelled annexin V and propidium iodide (PI). The cells that were transfected with either a pcDNA-ORF3 or an empty pcDNA3 plasmid were analyzed for apoptosis using annexin-V-FITC/PI double staining and subsequent flow cytometry at 24 and 48 h after transfection according to the manufacturer’s protocol. Approximately 1 × 106 cells were double stained with annexin-V-FITC and propidium iodide using the annexin-V-FITC Apoptosis Detection Kit (Sigma). Transfected cells were washed with cold PBS, resuspended in 1× binding buffer and combined with annexin-V-FITC and propidium iodide. The cells were incubated at room temperature in the dark for 15 min. Fluorescence was detected using a fluorescence-activated cell sorter (FACS Calibur, BD Biosciences). A total of 30,000 cells were collected for each sample and analyzed using CellQuest software (Becton Dickinson). A homogenous population of malignant cells or primary cells was chosen according to their size and granularity; debris and duplicates of cells were eliminated by gating. All treatments were performed at least in triplicate and all experiments were carried out three times.
Measurement of caspase activities
Caspase-3 and caspase-8 activities were measured first using a Caspase Colorimetric Assay Kit (Enzo) following the manufacturer’s instructions. In brief, ORF3-transfected B16F10 melanoma cells and WT mouse primary splenocytes were collected and lysed in chilled lysis buffer at 24 and 48 h post-transfection. The supernatant was removed, and the total protein concentration of each sample was quantified using Nanodrop™ spectrophotometer. Then, 150 μg of protein was diluted to 50 μl cell lysis buffer for each assay. 50 μl of 2× reaction buffer (containing 10 mM DTT) and 5 μl of the 4 mM IETD-pNA or DEVD-pNA (200 μM final concentration) substrate were added to each sample and incubated at 37 °C for 2 h. The optical densities were measured at 405 nm with an ELISA microtiter plate reader (Thermo Scientific Multiskan FC Microplate Photometer). The cells transfected with the empty pcDNA3 plasmid were used as negative control.
Caspase-3 activity was measured thereafter using intracellular staining in flow cytometry and an antibody that recognizes only cleaved and active form of Caspase-3. Since caspase-8 can be activated by autocleavage and can then trigger effector pro caspase-3 we determined the presence of a cleaved and active form of caspase-3 inside of B16F10 melanoma and PK15 cells.
Cell death in the presence of caspase inhibitors
To reveal any underlying caspase-independent pathways induced by PCV2 ORF3 Q-VD-Oph, an irreversible pan-caspase inhibitor was added to the cells prior to treatment. Mouse melanoma B16F10 or porcine kidney 15 (PK15) cells were pretreated with 5 μM Q-VD-Oph (quinolyl-valyl-O-methylaspartyl-[− 2, 6-difluorophenoxy]-methyl ketone) (Sigma) for 30 min to inhibit caspases and then transfected with PCV2-ORF3 or control plasmid as described before. Transfection efficiency, cell viability, and morphology were investigated as previously described. After 48 h cells were harvested and apoptosis was investigated by AnnexinV/PI staining as described above (in Flow Cytometry assay section).
Nuclear staining with 4′,6-diamidino-2-phenylindole
ORF3-transfected B16F10 cells grown on glass coverslips were washed 24 and 48 h post-transfection with PBS and subsequently fixed with 4% paraformaldehyde in PBS for 15 min. Fixed cells were then washed with PBS and incubated with 100 ng/ml DAPI (4′,6-diamidino-2-phenylindole; Sigma) in water for 30 min at room temperature in the dark. Stained slides were mounted using Fluoroshield Mounting Media (Sigma). The cells that were transfected with the empty plasmid were used as negative control. The nuclear morphology of the cells was examined by fluorescence microscopy. One hundred ORF3-positive and ORF3-negative control cells in mitotic phase were scored for normal/abnormal mitotic spindle formation.
Indirect immunofluorescence assay
For the indirect immunofluorescence assay, previously transfected and paraformaldehyde-fixed B16F10 cells were washed with PBS containing 0.1% saponin (PBS-S). Non-specific immunoreactions were blocked with PBS-S containing 1% bovine serum albumin (BSA; PAA) at room temperature for 40 min. The cells were then washed three times with PBS-S. Primary antibody, mouse anti-α-tubulin monoclonal antibody IgG (Dako) were diluted 1:1000 in PBS-S and incubated with cells at room temperature for 2 h. After washing with PBS-S, the cells were incubated with FITC-conjugated anti-mouse IgG (Dako) diluted 1:1000 in PBS-S at room temperature and protected from light for 1 h. After three further washes, nuclei were stained with DAPI as described above. The cells were visualized by fluorescence microscopy.
Co-expression of ORF3 and RGS16 in porcine peripheral blood mononuclear cells
Porcine peripheral blood mononuclear cells (poPBMC) were isolated from conventionally reared Yorkshire pig blood by density-gradient centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech) and cultivated directly in coverslip that was placed in the bottom of 24 well plates. Porcine PBMCs were cultured in RPMI 1640 medium (BioWhittaker) supplemented with 20 mM HEPES, 2 mM L-glutamine, 200 IU penicillin ml− 1, 100 μg streptomycin ml− 1, 0.5 μM 2-Mercaptoethanol and 5% FCS at 37 °C in a humidified incubator with 5% CO2 in 95% air. The cells were activated with LPS (lipopolysaccharide) from Escherichia coli serotype 0111: B6 (2,5 μg ml− 1; Sigma).
LPS-activated poPBMCs were then transiently transfected with pcDNA3.1-His-ORF3-mCherry in combination with pCEP-GFP-RGS16. The cells were seeded on glass coverslips placed in the bottom of six-well plates and transfected using FuGene® 6 reagent (Roche), following the manufacturer’s instructions. The cells were harvested 48 h after transfection.
The endogenous expression of RGS16 and ORF3 in poPBMCs was determined by indirect immunofluorescence assay using a rabbit-human RGS16-specific polyclonal antibody (Aviva Systems Biology) and a mouse monoclonal antibody recognizing the 6× His (Clontech) tag of the histidine-tagged ORF3 construct, respectively. Porcine PBMCs were fixed in 4% paraformaldehyde and non-specific immunoreactions were blocked by using PBS-S containing 1% BSA. After incubation with the primary antibody, the cells were stained with FITC-labeled antibody to rabbit Ig (Dako) or with monoclonal anti-mouse Igκ coupled to Texas red (Serotech). The cells’ nuclei were stained with the fluorescent dye Hoechst 33258 (Sigma). The cells were then visualized by fluorescence microscopy.
All fluorescence microscopy was performed using an Axioplan II Imaging fluorescence microscope equipped with appropriate filter sets, an Axiocam charge-coupled device camera and Axiovision software (Carl Zeiss Light Microscopy). Digital images were processed using Adobe Photoshop version CC software.
Subcutaneous grafts of B16F10-luc melanoma cells in RGS16 knockout mouse model
Exponentially growing B16F10-luc cells expressing stably firefly luciferase were harvested and injected subcutaneously into the right flank of 6–7-week-old female WT and RGS16 KO mice (1 × 106 cells per implant). Tumor development was monitored by luciferase activity using IVIS Lumina II imaging system (Caliper Life Sciences, now PerkinElmer). Before the scan, mice were shaved, injected intraperitoneally with D-luciferin (Regis Technologies, Illinois, USA) at 150 mg/kg body weight and dozed with 2.5% isoflurane. Sequential images were taken at 2 min intervals to determine the peak value of the luciferase signal. After 18 days mice were sacrificed, and the tumors were isolated and weighed.
In vivo induction and treatment of melanoma
For in vivo induction of melanoma, 8–10-week-old WT and RGS16 KO C57BL/6 mice were injected subcutaneously into the derma of the right side of the back with 5 × 104 B16F10 cells in 50 μl PBS. After tumor transplantation, its growth was followed at least every second day. After 18 days or with the appearance of either a dark pigmentation or a solid tumor on the melanoma inoculation site, 10 μg of pcDNA3-ORF3 expression plasmid (dissolved in 10 μl PBS) was injected into the tumor site. The control mice were injected with the same volume of the empty pcDNA3.1 plasmid. The length of the tumor was measured along the imaginary longitude of the back and its width was measured in the direction of the latitude with an accuracy of 0.5 mm using a digital caliper. Subcutaneous tumor volume was calculated using the following formula: V = π/6·ƒ·(length·width)3/2, where ƒ = 1.69 (+/− 0.3) for male mice and f = 1.58 (+/− 0.1) for female mice . Mice were sacrificed at day 28 after B16F10 melanoma cells injection, and all primary tumors were excised and weighed.
The statistical significance was determined using Student’s t-test. The significance level was set at a p-value of 0.05.