Immunohistochemical detection of the pro-apoptotic Bax∆2 protein in human tissues

Abstract

The pro-apoptotic Bax isoform BaxΔ2 was originally discovered in cancer patients with a microsatellite guanine deletion (G8 to G7). This deletion leads to an early stop codon; however, when combined with the alternative splicing of exon 2, the reading frame is restored allowing production of a full-length protein (BaxΔ2). Unlike the parental Baxα, BaxΔ2 triggers apoptosis through a non-mitochondrial pathway and the expression in human tissues was unknown. Here, we analyzed over 1000 tissue microarray samples from 13 different organs using immunohistochemistry. BaxΔ2-positive cells were detected in all examined organs at low rates (1-5%) and mainly scattered throughout the connective tissues. Surprisingly, over 70% of normal colon samples scored high for BaxΔ2-positive staining. Only 7% of malignant colon samples scored high, with most high-grade tumors being negative. A similar pattern was observed in most organs examined. We also showed that both Baxα and BaxΔ2 can co-exist in the same cells. Genotyping showed that the majority of BaxΔ2-positive normal tissues contain no G7 mutation, but an unexpected high rate of G9 was observed. Although the underlying mechanism remains to be explored, the inverse correlation of BaxΔ2 expression with tissue malignancy suggests that it may have a clinical implication in cancer development and treatment.

Detection of melanin-mediated false-positive for BaxΔ2 immunohistochemical staining in human skin tissues

“…We have previously shown that expression of BaxΔ2 protein in normal human tissues was very low: the BaxΔ2-positive cells were scattered in the connective tissues in a low rate (1-5%) [13]. However, the skin tissue was an exception.…”

“…Skin FFPE (Formalin-Fixed Paraffin-Embedded) tissue microarray slides were obtained from Biomax (Cat# T211a and SK481) containing both normal and tumor adjacent tissues. The BaxΔ2 monoclonal antibody was generated and validated previously [12], [13]. Anti-Cytokeratin 14 (CK-14) antibody was purchased from Abcam.…”

“…Expression of Bax∆2 in skin tissues appeared unique in comparison with other human tissues [13]. In addition to its typical distribution in the dermal connective tissue layer, most of the Bax∆2-positive cells lined up together at the bottom layer of epidermis [13]. Although the Bax∆2 antibody has been extensively validated and approved to be specific, we still wondered whether such strong positive staining is real Bax∆2 [12], [13].…”

“…Expression of BaxΔ2 in skin tissues appeared unique in comparison with other human tissues [13]. In addition to its typical distribution in the dermal connective tissue layer, most of the BaxΔ2-positive cells lined up together at the bottom layer of epidermis [13].…”

“…In addition to its typical distribution in the dermal connective tissue layer, most of the BaxΔ2-positive cells lined up together at the bottom layer of epidermis [13]. Although the BaxΔ2 antibody has been extensively validated and approved to be specific, we still wondered whether such strong positive staining is real BaxΔ2 [12], [13]. It has been shown that melanin from basal cells is a brown color comparable to the brown oxidized deposits in DAB staining, resulting in a potential false-positive in DAB-based staining [14].…”