Abstract
Background: Canine prostate cancer (PC) is a highly aggressive malignancy. However, in contrast to man, neither standard screening strategies nor curative therapeutic options exist for the companion animal. A prostate-specific membrane antigen (PSMA) screening as molecular marker akin to human PC is currently not available for dogs as data on specific canine PSMA detection are contradictory. Materials and Methods: To evaluate an antibody for specific canine PSMA detection by western blotting (WB), lysates of three canine prostatic cell lines (CT1258, DT08/40, DT08/46) were comparatively analyzed by WB and mass spectrometry (MS) to the human cell lines VCaP, LnCaP and PC-3. Results: MS analyses of the detected canine proteins confirmed cross reactivity of the antibody clone YPSMA-1 with canine PSMA. Conclusion: The MS analyses of the extracted canine protein bands proved that the YPSMA-1 clone is as well specific for canine PSMA in WB and, thus, represents a reliable tool for comparative PSMA studies.
Prostate cancer (PC) occurs spontaneously in man and dog. Although the incidence rate for the canine malignancy is less than 1% (1) it is often diagnosed at a very late stage (2). Canine PC is highly aggressive and metastasizes rapidly to other body parts, such as lymph nodes, lungs, liver, spleen and bones (3-5).
Consequently, reliable canine molecular markers for diagnosis and early detection are of need. However, in contrast to the situation in human medicine (6, 7), no screening methods are currently available for dogs.
As the canine prostate-specific membrane antigen (PSMA) protein presents a high sequence homology to the human homolog (8) and data on specific PSMA detection in pet dogs are contradictory (5, 9), we evaluated a monoclonal antibody raised against human PSMA for detection of the canine PSMA by western blot (WB) analysis and verified the detected proteins by mass spectrometry (MS).
Materials and Methods
Cell lines and culture conditions. The three canine cell lines derived from prostate tissue, CT1258 (adenocarcinoma), DT08/40 (transitional cell carcinoma) and DT08/46 (adenocarcinoma), were cultivated in medium 199 (Live Technologies GmbH, Darmstadt, Germany) supplemented with 10% of FBS superior (Biochrom AG, Berlin, Germany) 2% penicillin/streptomycin (Biochrom AG) and 29.3 ml/l NaHCO3 (Biochrom AG) in a humidified atmosphere, 5% CO2 at 37°C.
The three human prostatic cell lines LNCaP, PC-3 and VCaP were cultivated as recommended by the “Leibniz-Institut DSMZ GmbH” (Germany) under the same physical conditions as the canine cells.
Sample preparation. For protein analysis, equal cell numbers of the six cell lines were lysed using a buffer composed of 150 mM NaCl, 50 mM Tris, 1.0% Triton X-100, pH 8.0 (AppliChem GmbH, Darmstadt, Germany) and × 1 cOmplete Protease Inhibitor Cocktail Tablet (Roche, Mannheim, Germany) per 10 ml buffer.
To remove cell debris, the lysates were centrifuged at 10,000 × g and 4°C for 10 min. Supernatants were stored at −80°C.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis of denatured protein samples was performed in a discontinuous one-dimensional SDS polyacrylamide gel (SDS-PAGE) composed of a 5% stacking gel and a 12% resolving gel. The gel parts were prepared using 10% SDS and ammonium persulfate (AppliChem GmbH), 1.5 M Tris (AppliChem GmbH), 0.05% TEMED and 5% or 12% acrylamid/bisacrylamid (37.5:1) (AppliChem GmbH).
Twenty-eight μl of each of the six cell lysate supernatants were mixed with × 5 Laemmli buffer (1 M Tris, 20% β-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue and 50% glycerol (all chemicals from AppliChem GmbH), denatured at 95°C for 10 min and chilled on ice. Then, the protein samples were loaded together with a protein size standard marker (PageRuler; Thermo Scientific, Schwerte, Germany) on the prepared SDS-PA gels and separated. The electrophoresis was performed using a 1 × running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 20 mA for about 80 min at room temperature.
PSMA WB analysis on cell lysates. From left to the right: protein size standard PageRuler, human and canine cell lysates.
Western blot. After one-dimensional protein separation in a SDS-PAGE, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore KGaA, Darmstadt, Germany) via WB using a tank blotter (Bio Rad Laboratories GmbH, Munich, Germany). As the proteins were blotted at 350 mA for 35 min in 1x transfer buffer (2.9 g glycine, 5.8 g Tris, 200 ml methanol, adjusted to 1 l with H2O) the mixture was chilled with a cooling pack to avoid high heat dissipation.
Following blocking of the activated PVDF membrane in blocking buffer composed of TBS T (50 mM Tris, 150 mM NaCl and 0.01% Tween in H2O, pH 7.5; all chemicals from AppliChem GmbH) and 5% milk powder (AppliChem GmbH), the membrane was incubated overnight at 4°C with 10 μl/10 ml primary mouse monoclonal anti-PSMA antibody (YPSMA-1; Abcam, Cambridge, UK). Afterwards, the PVDF membrane was washed three times with TBS-T buffer and incubated in 2 μl/10 ml secondary antibody “anti-mouse IgG (H+L), AP Conjugate” (Promega, Mannheim, Germany). Finally, protein bands reactive with the PSMA antibody were visualized by membrane incubation in 10 ml alkaline phosphatase buffer (100 mM Tris, 100 mM NaCl, 5 mM MgCl2) supplemented with 100 μl NBT/BCIP substrate (Roche).
Target verification by mass spectrometric analysis. For verification of anti PMSA antibody cross-reactivity with the canine PSMA protein a SDS-PAGE was performed as described above. The proteins in the SDS-PAGE were visualized with Coomassie Brilliant Blue R-250 (Merck Millipore KGaA). The potential canine PSMA protein bands detected in the CT1258 lysate, showing a size of ≈50 kDa and ≈100 kDa, were recovered and used for protein verification by tryptic digest and liquid chromatography–MS analysis, as previously reported (10) (Figure 2a-b). The generated data were aligned to the canine PSMA protein sequence (GeneBank accession no. CCI39189.1) using Biotools MS proteomics software (Bruker Daltonic GmbH, Bremen, Germany).
The molecular mass of the targets was calculated using the software tool EditSeq (DNASTAR Lasergene Version: 7.1.0 (44), Madison, NY, USA).
Results and Discussion
PSMA detection in WB revealed, in accordance with prior reports, strong target expression in the human VCaP, LNCaP but low protein levels in PC-3 cells (11).
The expected size of the human PSMA homodimer separated by SDS-PAGE is approximately 100 kDa (12). The WB analysis of the human control samples revealed two prominent PSMA bands with a size of ≈50 kDa and ≈100 kDa (Figure 1). Interestingly, the canine CT1258 and DT08/40 lysates showed two potential PSMA protein bands as well matching the size of their human counterparts. In CT1258 and DT08/40, the corresponding PSMA expression was barely detectable, while being absent in DT08/46 cells (Figure 1).
(a and b). LC-MS analysis. PSMA bands were cut-out and proteins were in-gel digested; peptides were analyzed by LC-MS. Generated data were aligned to the canine PSMA protein sequence (GeneBank accession no. CCI39189.1) using the Biotools MS proteomics software. Identified peptides are indicated by grey bars and y and b ion series of MS/MS fragmentation are displayed by small red rectangles in these grey bars. The sequence coverage is given for both analyses in a) ≈100 kDa and b) ≈50 kDa protein bands.
The unexpected smaller PSMA band of ≈50 kDa could be a PSMA isoform or a degradation product. As the protein sequence partially covers the amino acids (aa) 72-758 of the canine PSMA (GeneBank accession no. CCI39189.1) (Figure 2 b), a degradation product would have a total length of 686 aa corresponding to a calculated molecular mass of ≈77 kDa. The observed product size in WB appears to be around 50 kDa. As the intact PSMA homodimer with a calculated mass of ≈87 kDa runs at ≈110 kDa in a SDS PAGE, a degradation product of ≈77 kDa would appear as well with a bigger molecular size in a SDS-PAGE. Owing to this, there is evidence that the smaller protein detected in WB in both species is a PSMA isoform, which additionally lacks some aa in between.
However, regardless of the origin of the smaller protein, the YPSMA-1 antibody clone presents cross-reactivity with the canine PSMA homolog in WB.
In a previous study, the same antibody clone was successfully used for immunohistochemical analysis in canine tissues but an evaluation for WB analysis and a verification of the specifity by MS was not focused (5).
Herein, specific reaction of the monoclonal mouse antibody clone YPSMA-1 raised against human PSMA was demonstrated with the canine protein orthologue in WB. The cross-reactivity with canine PSMA (GeneBank accession no. CCI39189.1) was confirmed by MS analyses of the recovered CT1258 protein bands (Figure 2 a-b).
In conclusion, the herein performed MS analyses of the extracted canine protein bands prove that the YPSMA-1 clone is specific for canine PSMA in WB, thus offering a reliable tool for on-going PSMA investigations in the pet dog.
Acknowledgements
The Authors would like to thank Dr. Saskia Willenbrock for proofreading the manuscript.
Footnotes
-
↵* These Authors contributed equally to this study.
- Received October 1, 2014.
- Revision received October 22, 2014.
- Accepted October 27, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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