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Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts
BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-ex...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684535/ https://www.ncbi.nlm.nih.gov/pubmed/23758893 http://dx.doi.org/10.1186/1746-6148-9-116 |
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author | Milovancev, Milan Hilgart-Martiszus, Ian McNamara, Michael J Goodall, Cheri P Seguin, Bernard Bracha, Shay Wickramasekara, Samanthi I |
author_facet | Milovancev, Milan Hilgart-Martiszus, Ian McNamara, Michael J Goodall, Cheri P Seguin, Bernard Bracha, Shay Wickramasekara, Samanthi I |
author_sort | Milovancev, Milan |
collection | PubMed |
description | BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. |
format | Online Article Text |
id | pubmed-3684535 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-36845352013-06-18 Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts Milovancev, Milan Hilgart-Martiszus, Ian McNamara, Michael J Goodall, Cheri P Seguin, Bernard Bracha, Shay Wickramasekara, Samanthi I BMC Vet Res Research Article BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. BioMed Central 2013-06-13 /pmc/articles/PMC3684535/ /pubmed/23758893 http://dx.doi.org/10.1186/1746-6148-9-116 Text en Copyright © 2013 Milovancev et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Milovancev, Milan Hilgart-Martiszus, Ian McNamara, Michael J Goodall, Cheri P Seguin, Bernard Bracha, Shay Wickramasekara, Samanthi I Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title | Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title_full | Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title_fullStr | Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title_full_unstemmed | Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title_short | Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
title_sort | comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684535/ https://www.ncbi.nlm.nih.gov/pubmed/23758893 http://dx.doi.org/10.1186/1746-6148-9-116 |
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