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Home , Programmed cell death

in vivo 18: 593-602 (2004)

Immunocytochemical Detection of Members of the Cascade of 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 and 3Department of Pediatrics, University of Southern California, Keck School of Medicine, Los Angeles, CA; 2Childrens Center for and Blood , 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 , University of Vienna, Vienna, Austria

Abstract. During the physiological process of PCD, the 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 . The family of is involved in the execution of genetically controlled PCD. Caspase-3 Apoptosis or programmed cell (PCD) is expressed in normal and neoplastically transformed 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 metazoans to control cell number in tissues and to eliminate (NuMA) and mediates the shrinkage and fragmentation of cell individual cells that threaten the ’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 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 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 () 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 (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 and this can be identified only in level of PCD is ensured after autocatalytic activation, experiments (6,7). processing a number of caspases and regulatory proteins, which strongly amplify the apoptotic process. The family of cysteine aspartyl 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 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 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 brain and neurons. than 100 various proteins belonging to diverse groups, such Consistent with a role for CPP32 in apoptotic cell death, clear as signaling, regulatory, structural, etc. The activities of differences in the relative intensity of CPP32 immunostaining caspase family members as part of the complex PCD were noted in some shorter-lived types of cells compared to process are regulated by a number of complex interactions longer-lived, including (a) germinal center (high) versus between them, adapter proteins and target substrates and mantle zone (low) B lymphocytes within the secondary different types of families (11-13). follicles of lymph nodes, spleen and tonsils; (b) mature The intrinsic part of the PCD program arises from the neutrophils (high) versus myeloid progenitor cells (low) in mitochondria or the so-called "killer " (14), because bone marrow; (c) corpus luteal cells (high) versus follicular

594 Bodey et al: Caspase-3, -6, -8 and -9 Expression in Childhood High-grade Astrocytomas: An Immunocytochemical Study granulosa cells (low) in the ovary; and (d) prostate secretory Caspase-8 (Mch5). The Fas/APO-1-receptor-associated epithelial cells (high) versus basal cells (low). CPP32 was also cysteine , caspase-8 or Mch5 (MACH/FLICE), is found to be highly expressed in atherosclerotic plaques and believed to be the enzyme responsible for activating a to be colocalized with apoptotic cells (18). protease cascade after Fas-receptor ligation, which leads to According to the literature, immunocytochemistry of cell death (25). Binding of caspase-8 to the caspases has only been carried out in neuroblastomas, but results in oligomerization of the caspase-8 protein, which in not in ASTRs. Frequently, neuroblastomas demonstrate turn drives its autoactivation through self-cleavage. Once spontaneous regression and cellular differentiation, which activated, the caspase-8 activates the downstream caspases, may at least partly be regulated by signaling done through committing the cell to undergo PCD. The Fas-apoptotic the nerve and its receptors, TRK-A and pathway is potently inhibited by the cowpox serpin CrmA, p75LNTR (19). Caspase-3 is actually required for the suggesting that Mch5 could be the target of this serpin. activation of four other caspases (-2, -6, -8 and -10) and also Bacterial expression of proMch5 generated a mature participates in a feedback amplification loop involving enzyme composed of two subunits, which are derived from caspase-9 (23). the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391 and Asp-401. Recombinant Mch5 is able to Caspase-6. The observation that the cell death process/activate all known ICE/Ced-3-like cysteine effector gene product Ced-3 is homologous to human proteases and is potently inhibited by CrmA. This contrasts interleukin-1beta-converting enzyme (caspase-1) has led to with the observation that Mch4, the second FADD-related the discovery of at least nine other human caspases, many cysteine protease that is also able to process/activate all of which are implicated as mediators of apoptosis (20-22). known ICE/Ced-3-like cysteine proteases, is poorly inhibited The activity of ICE-like proteases or caspases is essential for by CrmA. These data suggest that Mch5 is the most apoptosis (23). Multiple caspases participate in apoptosis in upstream protease that receives the activation signal from mammalian cells but how many caspases are involved and the Fas-receptor to initiate the apoptotic protease cascade what their relative contribution to cell death is poorly that leads to activation of ICE-like proteases (TX, ICE and understood. To identify caspases activated in apoptotic cells, ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, an approach to simultaneously detect multiple active Mch4 and Mch6) and the ICH-1 protease. On the other caspases was developed. Employing neoplastically hand, Mch4 could be a second upstream protease that is transformed cells as a model, it has been established that responsible for activation of the same protease cascade in CPP32 (caspase-3) and Mch2 (caspase-6) are the major CrmA-insensitive apoptotic pathways. active caspases in cells undergoing PCD, being activated in alpha (TNF-alpha) binding to the response to distinct apoptosis-inducing stimuli and in all cell TNF receptor (TNFR) potentially initiates apoptosis and lines analyzed. Both CPP32 and Mch2 are present in activates the transcription factor nuclear factor kappa B apoptotic cells as multiple active species. In a given cell line, (NF-kappaB), which suppresses PCD by an unknown these species remained the same irrespective of the mechanism (26). The activation of NF-kappaB was found to apoptotic stimulus used. However, the species of CPP32 and block the activation of caspase-8. The induction of apoptosis Mch2 detected varied between cell lines, indicating could, in fact, be prevented by treating cells with an differences in caspase processing. The influence of a inhibitor of caspase-8, implying a direct relationship number of environmental parameters, including pH, ionic between caspase-8 and apoptosis (27). strength, detergent and specific ion concentrations, on the activity and stability of four caspases involved in death Caspase-9. Caspase-9 was initially purified and identified as receptor-mediated apoptosis have been observed (22). the third protein factor, Apaf-3, that participates in caspase-3 As already alluded to, caspases play a major role in the activation in vitro. Procaspase-9 and Apaf-1 bind to each transduction of extracellular apoptotic signals and execution other via their respective NH2-terminal, CED-3 homologous of PCD in mammalian cells (24). Ectopic overexpression of domains in the presence of cytochrome c and dATP, an the short prodomain caspase-3 and -6 precursors in event that leads to caspase-9 activation. Activation of mammalian cells does not induce apoptosis, which is due to procaspase-9 by Apaf-1 in the cytochrome c / dATP- their inability to undergo autocatalytic processing/activation, dependent pathway requires proteolytic cleavage to produce suggesting that they depend on the long prodomain caspases the mature caspase-9 molecule (28). On the other hand, for activation. Since caspase-3 and -6 are the most deletion of Apaf-1’s WD-40 repeats leaves Apaf-1 downstream executioners of apoptosis, the constitutively constitutively active and capable of processing procaspase-9 active versions of these caspases could be used at very low independently of cytochrome c and dATP (29). Apaf-1- concentrations in gene therapy model systems to induce mediated processing of procaspase-9 occurs at Asp-315 by apoptosis in target tissues or neoplastically transformed cells. an intrinsic autocatalytic activity of procaspase-9 itself (30).

595 in vivo 18: 593-602 (2004)

Apaf-1 can form oligomers and may facilitate procaspase-9 invasiveness and a relatively low metastatic potential. autoactivation by oligomerizing its precursor molecules. Proteolytic enzymes, such as the cysteine proteinase cathepsin Once activated, caspase-9, also called initiator caspase, can S (CatS), have been implicated in the local invasion by start the caspase cascade involving the downstream neoplastically transformed astrocytoma cells resulting in activation of caspase-3, -6 and -7 PCD executioners. tumor recurrence even after the surgical resection (39). Depletion of caspase-9 from S-100 extracts diminished Flannery and co-workers’ immunohistochemical study caspase-3 activation. of the active site of caspase-9 revealed that the highest levels of CatS activity were present attenuated the activation of caspase-3 and cellular PCD in grade IV tumors. response in vivo, indicating that caspase-9 is the most The specific aims of this study were: 1) to identify the upstream member of the apoptotic protease cascade that is expression of caspase-3, -6, -8 and -9 in neoplastically triggered by cytochrome c and dATP. transformed astrocytic cells; 2) to identify the presence of Kuida and co-workers tested the hypothesis that caspase-9 caspase-3, -6, -8 and -9 in TIL, which thereby destroys the is a critical upstream activator of caspases through gene ’s anti-neoplastic tumor-infiltrating effector cells; and targeting in mice (31). The majority of caspase-9 knockout 3) to identify the intensity of apoptotic cell death within mice die perinatally with a markedly enlarged and high-grade ASTRs and glioblastomas. malformed cerebrum caused by reduced PCD during brain development. Caspase-9 deletion prevents activation of Materials and Methods caspase-3 in developing brain cells during ontogenesis in vivo and caspase-9-deficient cortical thymocytes show Tissues and tissue handling. In this immunohistochemical study, we resistance to a subset of apoptotic stimuli, including employed formalin-fixed, paraffin-wax- embedded tissue sections of human primary childhood anaplastic ASTRs (DAKO Corporation, absence of caspase-3-like cleavage and delayed DNA Carpinteria, CA, USA). The diagnoses of the specific subtypes were fragmentation. Moreover, the cytochrome c-mediated established according to WHO guidelines for the classification of cleavage of caspase-3 is absent in the cytosolic extracts of glioma by a clinical neurohistopathologist (40-44). Technical details caspase-9-deficient cells but is restored after addition of in of the immunohistochemical techniques used in this study have vitro translated caspase-9. Together, these results indicate already been elaborated by other investigators (45-47) and in the that caspase-9 is a critical upstream activator of the caspase studies published by our group (34, 48-52). cascade in vivo. Monoclonal antibodies ñ Anti-caspase-3 mouse monoclonal antibody was obtained from Childhood astrocytoma (ASTR). Labvision (Fremont, CA 94539, USA; Cat. # MS-1121). Recombinant full-length human caspase-3 protein was employed "This peculiarity of the membrane, namely, that it becomes as immunogen. Isotype: IgG2a. Clone: 3CSPO1, same as 7.1.44. continuous with the interstitial matter, the real cement, which The MoAB was purified from the ascites fluid by Protein A binds the nervous elements together and that in all its . properties it constitutes a tissue different from the other forms ñ Anti-caspase-6 mouse, anti-human monoclonal antibody (Cat. # of connective tissue, has induced me to give it a new name, MS-1126; Labvision). Recombinant full-length human caspase- 6 prodomain (34 kDa) was employed as immunogen. Ig Isotype/ that of neuro- (nerve cement)." - Rudolf Virchow (32) Light chain: IgG1/k. Clone: 6CSPO3, same as 14.1.190. The MoAB was purified from the ascites fluid by Protein G Malignant childhood ASTRs represent neuroectodermal chromatography. tumors appearing within the neuro-glial or macroglial central ñ Anti-caspase-8 mouse monoclonal antibody (Cat. # MS-1143- (CNS) (33). Gliomas can grow anywhere in R7, Labvision). Recombinant full length human caspase-8 the CNS, but in children they usually occur in the brain stem, protein was employed as immunogen (mol. weight of antigen: 55 the cerebrum, or the . The most common brain kDa). Ig Isotype: IgG1. Clone: 8 CSPO3 (same as 4.1.20). The MoAB was purified from the ascites fluid by Protein A tumors develop from glial cell precursors (astrocytes, chromatography. Immunoreactivity: cytoplasmic. oligodendrocytes and ependymocytes). Astrocytomas thus ñ Anti-caspase-9 mouse monoclonal antibody (Cat. # MS-1146- account for about 68 per cent of the primary brain tumors P2; Labvision). Recombinant full length human caspase-9 occurring in children younger than age 20 (34,35). protein was employed as immunogen (mol. weight of antigen: GBM, a grade IV astrocytoma in the World Health 46-48 kDa). Ig Isotype/light chain: IgG1/k. Clone: 9 CSPO3 Organization (WHO) classification, is a highly malignant and (same as 96.1.23). The MoAB was purified from the ascites fluid aggressive that is regarded as the prototype of by Protein A chromatography. Immunoreactivity: cytoplasmic. Staining of formalin-fixed and paraffin wax-embedded tissue neoplastic tissue capable of inducing neoangiogenesis (36,37). sections requires an antigen retrieval technique, employing The vascular of this tumor type represents a preliminary boiling in 10mM citrate buffer, pH 6.0 (NeoMarkers, hallmark for histopathological diagnosis (38). Furthermore, Cat. # AP-9003) for 10-20 minutes followed by cooling at room glial tumors are characterized by a high tendency to local temperature for 20 minutes.

596 Bodey et al: Caspase-3, -6, -8 and -9 Expression in Childhood High-grade Astrocytomas: An Immunocytochemical Study

Figure 1. Childhood anaplastic ASTR. Expression of caspase-3 in the cytoplasm of neoplastically transformed cells. Routine, 10% buffered formalin fixation. Paraffin-wax embedding. Alkaline phosphatase conjugated streptavidin-biotin antigen detection technique. Magnification: 200x.

Figure 2. Childhood glioblastoma. Presence of caspase-8 in the cytoplasm and some nuclei of neoplastic cells. Routine, 10% buffered formalin fixation. Paraffin-wax embedding. Alkaline phosphatase conjugated streptavidin-biotin antigen detection technique. Magnification: 200x.

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Antigen retrieval. No single antigen retrieval solution works well with Qualitative evaluation (61): (A) very intense red staining; (B) all antigenic epitopes and as such, directions from the antibody strong red staining; (C) light red staining; (D) negative staining. manufacturer should be followed. In this immunocytochemical research project, we employed the immunohistochemical method of Results "antigen liberation" or "antigen retrieval", when necessary (53-56). In the first step, antigen retrieval was sometimes achieved by single or combined enzymatic digestion (ficin, pepsin and trypsin from Zymed In this immunohistochemical study, the presence and cellular Labs., South San Francisco, CA, USA) prior to the primary antigen- localization of caspase-3, -6, -8 and -9 were observed in antibody reaction. Heat-induced epitope retrieval (HIER) (57,58), as anaplastic, high-grade ASTRs and in GBMs, employing a modified by us, was also employed. Antigen retrieval required sensitive, four-step, alkaline phosphatase conjugated antigen immersion of tissue sections in a Target Retrieval Solution (DAKO detection technique. The immunoreactivity demonstrated a Corporation) and heating in a water bath (95ÆC to 99ÆC). Unmasking dominant cytoplasmic pattern in more than 50 per cent of the of fixed antigen epitopes was also carried out by a single or combined tumor cells with high intensity staining (+++, A,B) for enzymatic digestion prior to the primary AA reaction. An increase in the quantity of detectable antigenic epitopes following HIER has caspase-3 and caspase-6 while a dominant cytoplasmic pattern been described for a number of antibodies. Our method using citrate- of caspase-8 and -9 was found in more than 10 per cent of the buffer (NeoMarkers, Union City, CA 94587, USA; Cat.# AP-9003) astrocytic brain tumor cells with high intensity staining (++, solution worked well, employing preliminary boiling in 10mM citrate A,B). In about 10 per cent of the neoplastic cells, caspase-3 buffer, pH 6.0 for 10-20 minutes followed by cooling at room (CPP32) (Figure 1) and caspase-6, and in about 1 to 5 per temperature for 20 minutes. Immunomorphological observations cent of the neoplastic cells, caspase-8 (Figure 2) and -9, were reported a significant increase in the intensity of immunostaining in translocated from the cytoplasm into the nuclei in regressing, response to this antigen retrieval technique. apoptotic cells. A similar antigen expression pattern was Immunohistochemical controls. In order to ensure the specificity of observed in TILs. The presence of active or mature caspase-8 the anti-caspase-3, -6, -8 and -9 antibodies used in this study, we and -9 in the tumor infiltrating TIL cells is indicative of a tested the immunoreactivity of several normal human control possible escape mechanism favorable for the further tumor tissues including: brain, adrenal, heart, stomach, small intestine, development and local invasion. large intestine, liver, kidney, pancreas, lung, testis, ovary, uterus, prostate, thyroid and spleen, all included in one checkerboard Discussion multitissue block (DAKO Corporation; code # T1065) (59,60). Several postnatal human thymic specimens were also used as negative and positive tissue controls. A number of neoplastically Our immunohistochemical study identified cytoplasmic transformed tissues, including malignant melanoma and lung expression of caspase-3 and -6 in more than 50 per cent of cancer tissues, represented the positive tissue controls. Additional tumor cells and caspase-8 and -9 in more than 10 per cent of controls for all tissues and MoABs included: 1) omission of the tumor cells in high-grade anaplastic ASTR and GBM. The primary, anti-caspase-3 MoAB; 2) utilization of only the enzymatic presence of caspases -3, -6, -8 and -9 were also detected in developer solution to detect the presence of endogenous alkaline the TILs, representing the host’s immune effector, mostly phosphatase activity; and 3) utilization of MOPC 21 mouse cytotoxic cells. myeloma IgG1 (ICN) as a replacement for the primary MoAB to determine non-specific myeloma protein binding to the antigen The immunohistochemical expression of caspases during epitopes of the screened tissues. progression was published in a variety of neoplastically transformed cells. For example, the presence Immunohistochemical evaluation. Qualitative and quantitative of caspase -3, -6 and -8 and their association to PCD was evaluation of the percent of antigen-positive cells and the intensity of observed in pre-neoplastic and neoplastic lesions of the caspase-3, -6, -8 and -9 immunostaining were conducted using a light breast (62). The observed material consisted of 9 benign microscope (Olympus, Japan) counting 100-200 cells from each of breast epithelial , 15 atypical hyperplasias, 74 in five to eight distinct areas in non-necrotic, ASTR, melanoma, lung situ and 82 invasive carcinomas. Increased caspase-3 adenocarcinoma and postnatal thymus tissues. Artifacts were avoided, while, on the other hand, morphologically characteristic areas were immunoreactivity, as compared to the staining of normal sought out. The presence of neoplastically transformed astrocytes and breast ductal epithelium, was seen in 22% of benign oligodendrocytes with heterogeneous IPs, the endothelial elements epithelial hyperplasias, 25% of atypical hyperplasias, 58% of small blood vessels, tumor infiltrating leukocytes and macrophages of in situ carcinomas and 90% of invasive carcinomas (the host's immunological effector cells) required careful qualitative (ductal or lobular or both). The corresponding percentages assessment. Non-vascular elements were also examined, but only for caspase -6 and -8 were 11%, 25%, 60%, 87% and 22%, morphologically distinct ASTR cells were scored. 57%, 84%, 83%, respectively. In high-grade in situ lesions, Quantitative evaluation (61): (++++) over 90% of the total cell number are positive; (+++) 50% to 90% of the total cell there were significantly more cases with strong caspase -3, number are positive; (++) 10% to 50% of the total cell number -6 and -8 immunoreactivity than in low- and intermediate- are positive; (+) 1% to 10% of the total cell number are positive; grade lesions (p=0.0045, p=0.049 and p=0.0001, (±) under 1% of the total cell number are positive; (–) negative. respectively). In invasive carcinomas, however, no

598 Bodey et al: Caspase-3, -6, -8 and -9 Expression in Childhood High-grade Astrocytomas: An Immunocytochemical Study association between a high tumor grade and caspase -3, -6 To identify the role of apoptosis in brain tumor cell death, or -8 expression was found (p=0.27, p=0.26 and p=0.69, the authors observed macromolecular (RNA and protein) respectively). The mean apoptotic index was 0.14±0.14% in synthesis and activity in the central to peripheral region of benign epithelial hyperplasias, 0.17±0.12% in atypical benign brain tumors derived from five patients who had not hyperplasias, 0.61±0.88% in in situ carcinomas and previously received radiotherapy or chemotherapy. Normal 0.94±1.21% in invasive carcinomas. In all cases, strong brain tissue (NBT) served as the control. RT-PCR analysis caspase -3, -6 and -8 positivity was significantly associated of tumor tissues covering central to peripheral regions with the extent of apoptosis (p<0.001, p=0.015 and detected mRNA overexpression of the pro-apoptotic gene p=0.050, respectively). The results demonstrated that bax in malignant tumors, indicating a commitment to synthesis of caspase -3, -6 and -8 is up-regulated in apoptosis. In the mitochondria-dependent death pathway, neoplastic breast epithelial cells in parallel to the increase caspase-9 and caspase-3 were overexpressed in tumors. The in the apoptotic index and progression of the breast lesions. increased caspase-3 activity cleaved poly(ADP-ribose) It is well established that caspase -8 and -10 act as initiator polymerase (PARP). Agarose gel electrophoresis detected caspases of the extrinsic apoptosis pathway, with caspase-9 a mixture of random and internucleosomal DNA as an initiator caspase of the intrinsic apoptosis pathway fragmentation in malignant brain tumors. Overexpression of (63). Caspase-3 is considered to be the main effector caspase pro-apoptotic bax, up-regulation of and caspase-3 involved in both intrinsic and extrinsic pathways. Alteration and occurrence of internucleosomal DNA fragmentation of apoptosis is essential for cancer development. Thus, were present, indicating that one mechanism of cell death analysis of the expression status of caspases, the main in malignant brain tumors is apoptosis and that the executioners of apoptosis, in cancer tissues is needed for a enhancement of this process therapeutically may promote sophisticated understanding of cancer biology. As such, we decreased tumor growth. explored caspase expression in brain tumors with the hope The goal of a recent study was to investigate whether that the results may be useful for future therapies. In a similar apoptosis occurs in T lymphocytes that invade (TIL) Fas study, Yoo and co-authors analyzed the expression of caspase- (FasL)-expressing GBMs and if its induction could 3, -8, -9 and -10 in 60 advanced gastric adenocarcinomas by be mediated by Fas (66). Apoptotic T lymphocytes were employing a tissue microarray detected in GBMs through the detection of cell-type approach. Immunopositivity was observed for caspase-3 in 57 markers combined with active caspase-3 immuno- (95%), caspase-8 in 56 (93%), caspase-9 in 54 (90%) and histochemical analysis, a recently introduced apoptosis- caspase-10 in 58 (97%) of the 60 . While 46 specific in situ ligation assay, as well as by examining adenocarcinomas (77%) expressed all of the caspases morphological criteria. Apoptotic T cells expressed Fas and examined, 14 cancers (23%) showed loss of expression in one were localized in the vicinity or in direct contact with FasL- or more caspases examined. Normal gastric mucosal cells expressing tumor cells. The T lymphocytes were undergoing showed none to weak expression of caspases 3, 8, 9 and 10. apoptosis in spite of Bcl-2 expression. Expression of Bax was Taken together, these results suggest that stomach cancer also detected in dying T cells, thus explaining the absence cells in vivo may need caspase expression for PCD. Also, of the protective effect of Bcl-2 because Bax inhibits Bcl-2 higher expression of the caspases in stomach cancer cells death-repressor activity. The results strongly suggested that than in normal gastric mucosal cells suggests that apoptosis GBM cells that express FasL can induce apoptosis in in susceptible stomach cancer cells might be easily triggered, invading immune cells. This phenomenon may play an thereby producing selective pressure to make more important role in these tumors' maintenance of immune apoptosis-resistant cells during tumor development. The privilege and evasion from immune attacks. Awareness of same could hold true for brain tumors. this phenomenon could be helpful in the development of The great majority of malignant glial tumors are incurable novel strategies for immunological treatment of malignant, with the current classical, three therapeutic modalities, high-grade ASTRs. including surgical resection, radiotherapy and chemotherapy Macrophages play an important role in the regulation and (64). This may well be the direct result of the biological fate of neoplastically transformed astrocytic cells (67). variability of these tumors, e.g. participation of possible multiple Although gliomas contain an abundance of macrophages, their stem cell lines in the histogenesis, intrinsic and acquired role in the apoptosis of gliomas is not well known. Chen and multidrug resistance. It is well established that low-grade co-workers, in an experimental protocol to treat glioma cells, ASTRs have an intrinsic tendency for progressive IP de- collected stimulated macrophages with lipo-polysaccharide and differentiation toward higher grade, more aggressive ASTRs. culture supernatants of activated macrophages. The results Cell death in the core of human brain tumors is triggered demonstrated that molecules released from activated by hypoxia and lack of , but the mode of cell death, macrophages significantly increased the apoptosis of glioma whether necrosis or apoptosis, is not clearly defined (65). cells via Fas/FasL and caspase-3 pathways. The level of soluble

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Fas did not appear to be involved in the mechanism dependent or independent pathway. Whether and how responsible for apoptosis seen in this study, as its level was apoptosis of glioma cells induced by activated macrophages barely detected in both experimental and control groups. Two is involved in these two pathways simultaneously are not cytokines, TNFalpha and IFNgamma, were significantly known. However, it is established that the alteration of elevated in the supernatant obtained from the activated molecules related to both death pathways led to PCD of macrophages. Considering the important role of these two glioma cells and the inhibition of xenograft glioma growth in molecules in the induction of apoptosis mediated by the mice. Apoptosis of glioma cells, induced by the activated Fas/FasL system, the present data suggest that TNFalpha and macrophage, is executed by way of both death receptor- IFNgamma were the main molecules to trigger the cascade of dependent and independent pathways and such an apoptotic reactions in glioma cells. Hence, the results indicated apoptosis-induced approach can effectively inhibit the that molecules released from the activated macrophages growth of glioma in vivo. As such, caspases can prove to be provide significant signals to stimulate the expression of highly beneficial in the development of future therapies that Fas/FasL and caspase-3, which function to induce apoptosis in trigger neoplastically transformed cell apoptosis. The recent glioma cells. article by Shinoura and co-workers discusses the novel Annonaceous acetogenins are a group of potential anti- strategies to induce PCD in glioma cells by transduction with neoplastic agents isolated from Annonaceae (68). In a adenoviral vectors carrying a variety of apoptosis-related recent study, annonacin, a cytotoxic mono-tetrahydrofuran , including , Fas, FADD, caspase-8, , acetogenin was purified from the seeds of Annona reticulata p33ING1, p73alpha, Bax, Apaf-1, caspase-9, IkappaBdN, and analyzed for its biological effects. It induced Bax caspase-3, Bcl-2 and Bcl-X(L) (71). The results suggested expression, enhanced caspase-3 activity and caused that adenoviral vector-mediated delivery of apoptosis-related apoptotic cell death in T24 bladder cancer cells. These genes other than p53 is a potentially useful gene therapy results suggest that annonacin is potentially a promising approach toward the treatment of human brain tumors. anti-neoplastic compound. Enhancing caspase activity should be further explored as a new treatment option in the References fight against cancer. 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