Comparative time-dependent analysis of potential inflammation biomarkers in lymphoma-bearing SJL mice



Monica H. Kristiansson,¹^§ Vadiraja B. Bhat,²^§ I. Ramesh Babu,³ John S. Wishnok,³ and Steven R. Tannenbaum^3,⁴^, J Proteome Res. 2007 May; 6(5): 1735–1744. ¹Division of Occupational and Environmental Medicine and Psychiatric, Epidemiology, University Hospital, 221 85 Lund, Sweden ²Division of Investigative Pathology, 1901 South First Street, Building 205, room 1R33, Temple, Texas 76504 ³ Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, room 56-731, Cambridge, MA-02139 ⁴Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, room 56-731, Cambridge, MA-02139 ^To whom correspondence should be addressed: Steven R. Tannenbaum, Underwood-Prescott Professor of Toxicology, Professor of Chemistry, Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731A, Cambridge, MA 02139, Phone: (617) 253-3729, Fax: (617) 252-1787, Email: srt@mit.edu ^§Both authors contributed equally to this work. Abstract SJL mice colonized with RcsX lymphoma cells undergo a rapid inflammatory response associated with biological and physiological effects including increased nitric oxide production and mutations in spleen DNA. By two weeks post-colonization, these changes were accompanied by both up- and down-regulation of a number of plasma proteins. In the experiments reported here, plasma from individual SJL mice were analyzed at several time-points over the two-week period in order to determine if there were sets of proteins whose expression varied in concert and thus might serve as early biomarkers for inflammation-related disorders. Samples were collected just prior to injection of the RcsX cells and then after 4, 8, and 12 days. Albumin and immunoglobulins were depleted and the samples were resolved by 1D gel electrophoresis. The gels were cut into 20 slices, and the proteins digested in-gel with trypsin. The digests were treated with iTRAQ reagents and then analyzed by LC/MS/MS. The resulting data were processed with two software packages, i.e., ProQuant and Spectrum Mill, and then subjected to K-means cluster analysis (K=4). The four clusters revealed a set of highly up-regulated proteins, a set of progressively up-regulated proteins, a set with no major changes, and a set that declined. The first cluster included haptoglobin, and serum amyloid A; the second included groups with several functions including protease inhibition, cell motility, and transport. The iTRAQ results for a selection of the up-regulated proteins, including haptoglobin, hemopexin, serum amyloid P component and ceruloplasmin, were confirmed with Western blots. Prominent down-regulated proteins included esterase-1, paraoxonase, and alpha-2-macroglobulin. Approximately 50% of the upregulated proteins are canonical acute phase proteins, while the remainder are regulated by the Nrf2 transcription factor. Keywords: inflammation, SJL mouse, lymphoma, iTRAQ, tumor progression, biomarker, cluster analysis, acute phase proteins

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