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Assessment of Apoptosis by Immunohistochemical Markers Compared to Cellular Morphology in Ex Vivo–stressed Colonic Mucosa

Hana Holubec, Claire M. Payne, Harris Bernstein, Katerina Dvorakova, Carol Bernstein, Caroline N. Waltmire, James A. Warneke and Harinder Garewal

Department of Microbiology–Immunology (HH,CMP,HB,KD,CB,CNW), and Department of Surgery (JAW), College of Medicine, University of Arizona, Tucson, Arizona; and Section of Hematology/Oncology, Tucson Veteran Affairs Medical Center, Tucson, Arizona (HG)

Correspondence to: Carol Bernstein, Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724. E-mail: bernstein3{at}earthlink.net

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (H2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and H2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

(J Histochem Cytochem 53:229–235, 2005)

Key Words: apoptosis • bile acid • deoxycholate • colonic mucosa • caspase-3 • cytokeratin-18 • lamin A • histone H2AX

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