Evaluation of S100A10, annexin II and B-FABP expression as markers for renal cell carcinoma

Abstract

This study aimed to analyze expression of S100A10, annexin II and B-FABP genes in renal cell carcinoma (RCC) and their potential value as tumor markers. Furthermore, any correlation between the gene expression and prognostic indicators of RCC was analyzed. Expression of each gene was estimated by RT-PCR in the non-neoplastic (normal) and tumorous parts of resected kidney samples. Also, each antigen was immunostained in RCC and normal kidney tissues. Expression of the S100A10 gene averaged 2.5-fold higher in the tumor than that in the normal tissues (n = 47), after standardization against that of beta-actin. However, expression of annexin II, a natural ligand of S100A10, was only 1.64-fold higher. In the tissue sections of RCC, S100A10 and annexin II were immunostained in membranes. In the normal renal epithelia, however, both antigens were stained in the Bowman's capsule and the tubules from Henle's loop through the collecting duct system, but not in the proximal tubules, from where most RCC are derived. In contrast, expression of the B-FABP gene was 20-fold higher in the tumor. No B-FABP was immunohistochemically detected in normal kidney sections, but it was stained in the cytoplasm of RCC tissue sections. S100A10 and B-FABP genes were overexpressed regardless of nuclear grade and stage of RCC. Immunopositivity in RCC tissues (n = 13) was 100% for S100A10 and annexin II, and 70% for B-FABP; however, no clear relationship was observed in either antigen with nuclear grade and stage. It was found that all three performed well as RCC markers. B-FABP was most specific to RCC, as it was expressed little in normal kidney tissues.

Figure 1

Transcriptional expression of (A) S100A10, (B) annexin II and (C) B‐FABP genes in renal cell carcinoma. The arbitrary unit represents the PCR‐amplified expression of the gene plotted against that of the β‐actin gene in the normal and tumor parts of surgically resected kidney samples (n = 47). N, normal, T, tumor.

Figure 2

Immunohistochemistry for the three antigens on the serial sections of cancerous and normal renal tissues. (A–C) Serial sections of G2 renal cell carcinoma (original magnification ×14). Positive staining for S100A10 (A) and annexin II (B) is diffusely found on the plasma membrane and occasionally appeared faintly in the cytoplasm. Immunopositivity of B‐FABP (C) is seen in the cytoplasm of the carcinoma cells. (D, E) Normal renal cortex (original magnification ×14). Bowman's capsule and some of the distal convoluted tubules (asterisks) and glomerular podocytes (arrows) are positively stained for S100A10 (D) and annexin II (E). Immunostaining is also found in endothelial cells (arrowheads). The few proximal tubules (PT) represent the signals. (F, G) Normal renal medulla (original magnification ×14). The immunopositivity for S100A10 (F) is in agreement with that for annexin II (G). Positive reactions observed are mainly associated with the plasma membrane of the collecting duct (asterisks) and the thin portion of Henle's loop (arrows). Endothelium (arrowheads) is also diffusely positive for both proteins.

Figure 3

Localization of S100A10 and annexin II complex on the membrane of cultured renal cell carcinoma cells. (A) Pull‐down assay for S100A10 and annexin II complex. S100A10 and annexin II were analyzed in the lysate from membrane (m) and cytosol (c) fraction of TUHR14TKB and RCC10RGB9 cells (a) by western blotting or (b) after being pulled down with mAb S100A10–Sepharose beads. M indicates the marker for S100A10 and annexin II. (B) Immunofluorescent detection of (a) pre‐immune serum, (b) S100A10 and (c) annexin II on RCC10RGB9 cells. Cells were stained without being made permeable with NP‐40.