Characterization of the 12C4 survivin monoclonal antibody and insight into the expression of survivin in human adult tissues

Abstract

Aims: To elucidate the expression and regulation of survivin in normal tissues.

Methods and results: A novel monoclonal antibody (12C4) to survivin was generated. Application of this antibody to determine survivin expression in human normal adult tissues revealed that most adult tissues do not express survivin and, where it is present, survivin is largely restricted to a small subset of epithelial cells and cells with proliferative potential such as thymus. Survivin expression among positive tissues showed individual variations, ranging from zero to < 5% positive cells in epithelial cell populations. Testis is the only human adult tissue highly expressing survivin, with 60-70% positivity in the nuclei of spermatogonia. Consistent with deregulated expression of survivin associated with oncogenesis, we found that certain ligands and transcription factors differentially modulate survivin promoter activity in cancer cells versus normal/untransformed cells.

Conclusion: Deregulation of survivin transcription controls in individual epithelial cells may contribute to oncogenesis in various human adult tissues.

Figure 1

Characterization of the anti-survivin monoclonal antibody, 12C4. a, Diagram of the glutathionine-S-transferase (GST)-fused survivin proteins with and without various truncations. Generation of the corresponding vectors for expressing these proteins is described in Materials and methods. b, The first 27 amino acids of the survivin protein sequence are essential for recognition by clone 12C4. The 12C4 antibody was characterized by Western blot analysis (left panel; note that top bands are survivin and low bands are degraded) using various truncated survivin proteins as indicated at the top. Protein expression was confirmed by Western blotting with anti-GST antibodies (middle panel), and protein loading was visualized by Coomassie blue staining (right panel).

Figure 2

Specificity of mAb clone 12C4 to various survivin variants/isoforms. a, Diagram of protein structures for the different survivin variants. b, Clone 12C4 recognizes all survivin isoforms with similar affinity. HeLa cells were transfected with pEGFPc1 empty vector or with various survivin variants as shown. Expression of enhanced green fluorescence protein (EGFP)-fused survivin variants was determined by Western blots using the clone 12C4 (note: top bands are survivin variants, and low bands are degraded). c, Clone 12C4 specifically recognizes endogenous survivin expression in various cancer cell lines. Survivin expression in lysates from various normal and cancer cell lines was determined using the 12C4 survivin mAb and the survivin polyclonal antibody from Santa Cruz (sc-10811) by Western blots, respectively. Actin is an internal control for protein loading. MCF-7, a breast cancer cell line; HCT-116, a colon cancer cell line; A549, a lung cancer cell line; HeLa, a cervical carcinoma cell line; NIH3T3, a mouse embryo fibroblast cell line which is known with very low or no survivin expression; HDF, a primary normal human dermal fibroblast cell line, which was also shown to be survivin negative.

Figure 3

Images from the immunohistochemical study of survivin expression in human adult tissue. Survivin is highly expressed in human testis (a). Survivin is expressed in a limited subset of epithelial cells in the intestinal crypts (b) and basal layers of squamous epithelia (c,d). Left panels in a and b are negative controls processed with an IgG2a subclass mouse mAb (negative control reagent).

Figure 4

Different modulation of survivin promoter activity by vitamin D3 (VD3). a, VD3 down-regulates survivin promoter activity in MCF-7E breast cancer cells (charcoal dextrin-stripped medium). MCF-7E cells were transfected with the survivin promoter-luciferase reporter construct, pLuc-2840, and treated with and without VD3 for 48 h before the luciferase assay. Luciferase activity was normalized to Renilla luciferase (internal control) activity as arbitrary units and plotted as histograms. Each bar represents the mean ± SD derived from three independent assays. b, VD3 has no inhibitory effect on survivin promoter activity in NIH3T3 fibroblast cells. The experimental conditions were the same as in a. Each bar represents the mean ± SD derived from three independent assays.

Figure 5

Different modulation of survivin promoter activity in cancerous versus non-transformed cells by AP-2 family transcriptional factors. The survivin promoter-luciferase construct, pLuc-2840, was transfected into various cell lines together with pRSV empty vector or one of the AP-2 expression vectors (α, β, γ) as shown. Cells were lysed for luciferase reporter assay 36–48 h after transfection. Luciferase activity was normalized to Renilla luciferase (internal control) activity as arbitrary units and plotted as histograms. Representative experiments are shown. Each bar represent the means ± SD from two independent experiments.