Journal of Pathology J Pathol 2004; 204: 175–182 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1630 Original Paper Caspase-3 expression is reduced, in the absence of cleavage, in terminally differentiated normal oral epithelium but is increased in oral squamous cell carcinomas and correlates with tumour stage Angela Hague, 1 * John W Eveson, 1 Marion MacFarlane, 2 Suzanne Huntley, 1 Nari Janghra 1 and Selvam Thavaraj 1 1 Department of Oral and Dental Science, University of Bristol Dental School, Lower Maudlin Street, Bristol, BS1 2LY, UK 2 MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 136, Leicester Road, Leicester, LE1 9HN, UK *Correspondence to: Dr Angela Hague, Department of Oral and Dental Science, University of Bristol Dental School, Lower Maudlin Street, Bristol, BS1 2LY, UK. E-mail: a.hague@bristol.ac.uk Received: 14 May 2004 Revised: 30 June 2004 Accepted: 4 July 2004 Abstract Oral carcinomas are known to have a greater apoptotic index than normal oral epithelium, evident as shrinking cells with condensed chromatin. In this study, these morphologically apoptotic cells stained positively for cleaved (active) caspase-3. In normal oral epithelium, cleaved caspase-3 positive-cells were only rarely detected. The terminally differentiated surface epithelial layers did not express cleaved caspase-3. The caspase-3 pro-enzyme showed a gradient of expression in normal oral epithelium, decreasing with differentiation. No expression was detectable in surface epithelial layers. Lack of expression of the major ‘executioner’ caspase-3 may, at least in part, explain differences in morphology between terminally differentiated and apoptotic cells. In cancers of different tissue origins, caspase-3 pro-enzyme expression can be either increased or decreased compared with normal tissue counterparts. To determine how caspase-3 expression alters during oral carcinogenesis, caspase-3 expression was compared in 39 samples of normal oral epithelium and 54 oral squamous cell carcinomas. Squamous cell carcinomas had more intense caspase-3 staining than normal epithelium (p < 0.001). Moreover, within the oral squamous cell carcinoma series, there was significantly more intense nuclear and cytoplasmic staining with increasing STNMP stage (p = 0.017 and 0.03, respectively). This was a reflection of significant associations with site (S), palpable lymph nodes (N), and differentiation (P). Both caspase-3 staining intensity and the percentage of cells positive for caspase-3 were inversely associated with differentiation. Studies of the mechanisms by which high levels of caspase-3 expression are tolerated in oral carcinoma cells may identify targets that can be used to harness caspase-3 overexpression for therapeutic benefit. Copyright  2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: caspase-3; apoptosis; terminal differentiation; oral epithelium; oral carcinoma Introduction The caspases are a family of cysteine proteases involved in signalling and execution of apoptotic cell death. They are synthesized as pro-enzymes and activated by proteolytic cleavage. Activated caspases cleave target proteins after aspartic acid residues and may cleave other caspases as part of the apoptosis- signalling cascade. Downstream or ‘effector’ caspases cleave specific molecular targets such as DNA repair enzymes, for example poly(ADP-ribose)-polymerase (PARP), ICAD (an inhibitor of the apoptotic endonu- clease CAD), regulators of the cell cycle, such as Rb and MDM2, and proteins involved in cellular struc- ture, cell–cell and cell–matrix adhesion. Caspase-3 is a major effector caspase, activated by cleavage to pro- duce 12 kD and 17 kD subunits, which combine to form a heterotetrameric enzyme. The pro-enzyme is expressed in a wide range of tissues, including those with a high tissue turnover such as intestinal epithe- lium and epidermis [1]. In secondary lymphoid fol- licles, caspase-3 is strongly expressed in short-lived germinal centre B cells, but is absent from longer- lived mantle zone B cells [1]. Caspase-3 is expressed in tissues with a greater propensity for apoptosis and the consequences of caspase-3 expression on apoptosis are tissue- and stimulus-specific [2]. Studies of caspase-3 expression in cancers have begun to show differences specific to tumour type. Caspase-3 expression is reduced compared with cor- responding normal tissues in Hodgkin’s lymphoma [3], hepatocellular carcinoma [4], and prostate carci- noma [5]. Moreover, in prostate carcinogenesis, there is significant loss of expression in high-grade tumours compared with benign hyperplasia [6,7]. By contrast, increased caspase-3 expression has been demonstrated Copyright  2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.