An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer - PubMed (original) (raw)

Comparative Study

An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer

F M Brichory et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. The purpose of this study was to identify proteins that commonly induce a humoral response in lung cancer by using a proteomic approach and to investigate biological processes that may be associated with the development of autoantibodies. Aliquots of solubilized proteins from a lung adenocarcinoma cell line (A549) and from lung tumors were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for primary antibodies. Sera from 54 newly diagnosed patients with lung cancer and 60 patients with other cancers and from 61 noncancer controls were analyzed. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with squamous cell lung carcinoma but none of the noncancer controls exhibited IgG-based reactivity against proteins identified as glycosylated annexins I and/or II. Immunohistochemical analysis showed that annexin I was expressed diffusely in neoplastic cells in lung tumor tissues, whereas annexin II was predominant at the cell surface. Interestingly, IL-6 levels were significantly higher in sera of antibody-positive lung cancer patients compared with antibody-negative patients and controls. We conclude that an immune response manifested by annexins I and II autoantibodies occurs commonly in lung cancer and is associated with high circulating levels of an inflammatory cytokine. The proteomic approach we have implemented has utility for the development of serum-based assays for cancer diagnosis as we report in this paper on the discovery of antiannexins I and/or II in sera from patients with lung cancer.

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Figures

Figure 1

Silver staining of A549 lung adenocarcinoma cell proteins separated by 2-D PAGE. Arrows point to the location of annexins I, II, IV, and V (A1, A2, A4, and A5) spots in the pattern.

Figure 2

(A) IgG1-based reactivity against annexin II protein in a Western blot of A549 proteins, using a lung cancer patient serum. (B) Close-up of a Western blot showing reactivity with antiannexin II mAb, confirming identity of the reactive protein shown in A. (C) Matrix-assisted laser desorption ionization–time of flight spectra obtained from protein A2 after trypsin digestion and tryptic peptide sequences from annexin II matching with peaks obtained from the spectra.

Figure 3

(A) IgG1-based reactivity against both annexin I and II proteins by using a patient serum. (B) Close-up of a Western blot showing reactivity of annexin I protein with antiannexin I mAb. (C) Mass spectrometry identification of annexin I after trypsin digestion of the protein A1.

Figure 4

Immunohistochemical analysis of annexin I and II expression in lung carcinomas. Representative immunoreactivity of annexins I and II in lung adenocarcinoma (ACA) and squamous cell carcinoma (SCC) (×300). Annexin I staining showed a mixture of nuclear, cytoplasmic, and membranous immunoreactivity, whereas annexin II staining showed immunoreactivity localized to the cytoplasmic membrane. T and S denote tumor and stroma tissue, respectively.

Figure 5

(A) Western blot analysis of purified annexin I before enzymatic treatment. (B) Western blot analysis of purified annexin I after endoglycosidase F treatment, showing a basic shift in annexin I migration. A and B were 2-D PAGE Western Blot-hybridized with antiannexin I mAb. (C) One-dimensional SDS/PAGE Western blot of purified annexin I, hybridized with a serum from an annexin I antibody-positive lung cancer patient. Hybridization was observed before (− endo F) but not after (+ endo F) N-deglycosylation by endoglycosidase F.

Figure 6

Serum IL-6 levels in annexin autoantibody (autoAb)-positive and autoAb-negative lung cancer patients, in healthy subjects, and in patients with chronic lung disease.

Figure 7

Western blot analysis of proteins from the A549 cells, treated or untreated with IL-6, separated by one-dimensional SDS/PAGE, and subsequently hybridized with either antiannexin I, antiannexin II, or anti-α-tubulin (control) antibodies. An increase in the membrane-associated fraction (MAP) was observed after IL-6 treatment. SP and CPE denote secreted protein and cytosolic protein extract fractions, respectively.

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An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer - PubMed (original) (raw)