Comparison of immunohistochemistry for activated caspase‐3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC‐3 subcutaneous xenografts

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Abstract
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique has been extensively used for the detection and quantification of apoptosis in histological tissue sections. However, the interpretation and specificity of this assay have been controversial. With accumulating knowledge of the molecular mechanisms of cell death and the discovery of the caspases as key mediators of apoptosis, more direct and earlier measurements of apoptosis in tissue sections have emerged. This study, using antibodies that specifically recognize activated caspase-3 and caspase-cleaved cytokeratin (CK) 18, evaluated whether immunohistochemical stains would improve the detection and quantification of apoptosis in tissue sections, compared with the TUNEL assay. Tumour xenografts of the prostate cancer cell line PC-3 were used as an example, since these tissues contain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were calculated by computer-assisted image analysis following identification of apoptotic cells by morphological analysis, the TUNEL assay, activated caspase-3 and cleaved CK18 immunohistochemistry. The results indicated that activated caspase-3 immunohistochemistry was an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. An excellent correlation (R = 0.89) between the apoptotic indices obtained using activated caspase-3 and cleaved CK18 immunostaining was observed. A good correlation (R = 0.75) between the apoptotic indices obtained using activated caspase-3 immunostaining and the TUNEL assay was also found. Activated caspase-3 immunohistochemistry is therefore recommended for the detection and quantification of apoptosis in tissue sections.