in vivo 18: 593-602 (2004) Immunocytochemical Detection of Members of the Caspase Cascade of Apoptosis in High-grade Astrocytomas BELA BODEY1,2, VIVIAN BODEY1,2, STUART E. SIEGEL2,3, AEJAZ NASIR4, DOMENICO COPPOLA4, ARDESHIR HAKAM4 and HANS E. KAISER5 1Department of Pathology and 3Department of Pediatrics, University of Southern California, Keck School of Medicine, Los Angeles, CA; 2Childrens Center for Cancer and Blood Diseases, Childrens Hospital Los Angeles, Los Angeles, CA; 4H. Lee Moffitt Cancer Center (MCC) & Research Institute, University of South Florida, Tampa, FL; 5Department of Pathology, School of Medicine, University of Maryland, Baltimore, MD, U.S.A. and Department of General and Experimental Pathology, University of Vienna, Vienna, Austria Abstract. During the physiological process of PCD, the cell the apoptotic cells. This phenomenon may play an important role initiates a sequence of events culminating in the disintegration of in these tumors' maintenance of immune privilege and evasion of the cell into small, membrane-bound apoptotic bodies. The immune attacks. We suggest that caspase-3, -6, -8 and -9 intrinsic part of the PCD program arises from the mitochondria immunocytochemistry could have prognostic and immuno- when it releases cytochrome c from the mitochondrial therapeutic significance in the treatment of these highly malignant intermembrane space into the cytosol, forming the caspase- glial tumors. activating complex or apoptosome. The family of caspases is involved in the execution of genetically controlled PCD. Caspase-3 Apoptosis or programmed cell death (PCD) is expressed in normal and neoplastically transformed human cells and, like other caspases, is synthesized as an inactive, 32kDa "Apoptosis is a cell suicide mechanism that enables proenzyme. Caspase-6 cleaves nuclear mitotic apparatus protein metazoans to control cell number in tissues and to eliminate (NuMA) and mediates the shrinkage and fragmentation of cell individual cells that threaten the animal’s survival. Certain nuclei. Caspase-8 is an initiation caspase that activates the cells have unique sensors, termed death receptors, on their caspase cascade during apoptosis, while caspase-9 is the initiator surface. Death receptors detect the presence of extracellular caspase in the caspase cascade in apoptotic normal and death signals and, in response, they rapidly ignite the cell’s neoplastically transformed cells. During our immunocytochemical intrinsic apoptosis machinery" (1). study, a sensitive, four-step, alkaline phosphatase conjugated Normal development (pre- and postnatal), cell maturation antigen detection technique was employed. The results did in fact and differentiation in a multicellular mammalian organism, demonstrate the presence of high apoptotic activity within the as well as the maintenance of cellular homeostasis, require cellular microenvironment of high-grade astrocytomas and a perfect, dynamic co-regulation of cell proliferation and cell glioblastomas. The observations identified cytoplasmic expression death (2,3). Furthermore, the balance between cell of caspase-3 and caspase-6 in more than 50 per cent of tumor proliferation and cell death determines the growth and cells, caspase-8 and caspase-9 in more than 10 per cent of tumor differentiation of every complex multicellular tissue. Three cells in high-grade anaplastic ASTR and glioblastoma. The decades ago, Kerr and co-authors proposed the introduction immunocytochemical expression pattern in about 10 per cent of of the scientific term "apoptosis" from the Greek apo (away the tumor cells for caspase-3 and caspase-6 and about 1 to 5 per from) and ptosis (falling) for the morphological cent of the tumor cells for caspase-8 and caspase-9 demonstrated nomenclature (4). Apoptosis is thus described as a process a translocation tendency from the cytoplasm to the cell nuclei in of leaves falling from the trees or the shedding of petals from flowers. It is a form of suicidal cell death that requires the active cellular synthesis of molecules involved in processes that provide the energy necessary for programmed cell death Correspondence to: Bela Bodey, M.D., D.Sc., 8000-1 Canby Avenue, Reseda, CA 91335, U.S.A. e-mail: [email protected] (PCD) to occur. PCD is a distinct mode of cellular suicide and represents one of the most important regulatory Key Words: Antigen detection technique, caspases, apoptosis, mechanisms of homeostasis, because it eliminates abundant astrocytoma, glioblastoma. and unwanted cells (5). 0258-851X/2004 $2.00+.40 593 in vivo 18: 593-602 (2004) Specifically, apoptosis is a physiological process wherein it is the release of cytochrome c from the mitochondrial the cell initiates a sequence of events culminating in the intermembrane space into the cytosol that develops into the fragmentation of its DNA, nuclear collapse and, finally, caspase-activating complex or apoptosome (15). The disintegration of the cell into small, membrane-bound apoptosome is completed from cytochrome c, ATP, Apaf-1 apoptotic bodies. Dramatic changes in the morphological and caspase-9 zymogen, the last two of which interact via their structure of cells such as: 1) condensation of the nuclear CARDs. Smac (or DIABLO) is a recently identified, novel (chromatin) and cytoplasmic structures (especially the proapoptotic molecule that is also released from mitochondria mitochondria); 2) blebbing of the cell membrane; 3) into the cytosol during apoptosis. Smac functions by characteristic swelling of the endoplasmic reticulum; and 4) eliminating the caspase-inhibitory properties of the inhibitors fragmentation of the cells in membrane-bound apoptotic of apoptosis proteins (IAP), particularly XIAP (16). After the bodies, are the signs of total cell destruction. Complete initial caspase-9 activation and the activation of the following breakdown of the cell membrane and cytoplasm is effector caspases, the so-called terminal caspase-cascade is associated with the advanced stage of apoptotic changes, built up, a two-step process that is essential to PCD. The high during secondary necrosis and this can be identified only in level of PCD is ensured after autocatalytic activation, in vitro experiments (6,7). processing a number of caspases and regulatory proteins, which strongly amplify the apoptotic process. The family of cysteine aspartyl proteases or caspases. Caspases are the executioners in the apoptotic or programmed cell death Caspase-3. Caspase-3 is ubiquitously expressed in human cells (PCD) pathway (8). Molecular biological research revealed and, like other caspases, is synthesized as an inactive, 32 kDa that the caspase family functions as a well-directed orchestra proenzyme. Upon activation, caspase-3 is cleaved at Asp28- of at least 14 proteases, all containing a common active site of Ser29 and Asp175-Ser176, thereby generating two subunits of cysteine within the conserved peptide sequence QACXG. 17 kDa and 12 kDa, respectively. Activation of caspase-3 Their cleavage target is the peptide bond C-terminal to occurs in response to a wide variety of PCD inducers asparate residues. Caspases are divided into three subgroups including Fas-FasR. The in vivo patterns of CPP32 (caspase-3) upon their tetrapeptide sequence recognition: Group I gene expression were determined by Krajewska and co- caspases (caspase-1, caspase-4 and caspase-5) generally prefer workers employing an immunohistochemical approach and the (W/L)EHD sequence; Group II caspases (caspase-3 and formalin-fixed, paraffin wax-embedded normal human tissues caspase-7) prefer DEXD; and Group III caspases (caspase-6, (17). Krajewska and co-workers used a rabbit polyclonal caspase-8, caspase-9 and caspase-10) recognize (L/V)EXD. antiserum against recombinant human CPP32 protein, which The VAD tripeptide is targeted by all caspases; therefore, was proven specific by immunoblot analysis of various human VAD is the structural base for pan-caspase reagents (9). tissues and cell lines. CPP32 immunoreactivity was selectively All caspases are present in mammalian cells in the form found in certain cell types and was typically present within of enzymatically inert zymogens (named procaspases). The the cytosol, although occasional cells also contained nuclear zymogens undergo cleavage, resulting in four large and immunostaining. CPP32-positive normal cells included small subunits. These active or mature caspases represent a epidermal keratinocyes, cartilage chondrocytes, bone functional tetramer containing two distinguished active sites osteocytes, heart myocardiocytes, vascular smooth muscle and two heterodimer subunits, one 20 kD (p20) and an cells, bronchial epithelium, hepatocytes, thymocytes, plasma other 10 kD (p10) (10). The prodomain is typically short for cells, renal tubule epithelium, spermatogonia, prostatic the so-called effector caspases (caspase-3 and caspase-7) secretory epithelial cells, uterine endometrium and and longer for the rest of them. The longer prodomain can myometrium, mammary ductal epithelial cells and the include recognizable motifs, such as the CARD (caspase gastrointestinal epithelium of the stomach, intestine and activation and recruitment domain: in caspase-1, caspase-2, colon. In contrast, weak or absent CPP32 immunoreactivity caspase-4 and caspase-9) and the DED (death effector was observed in endothelial cells, alveolar pneumocytes, domain: in caspase-8 and caspase-10). kidney glomeruli, mammary myoepithelial cells, Schwann Recently, it has been shown that caspases target more cells and most types of