Journal of Histochemistry and Cytochemistry, Vol. 49, 1269-1276, October 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Expression of Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain, a Pyrin N-terminal Homology Domain-containing Protein, in Normal Human Tissues

Junya Masumotoa,c,d, Shun'ichiro Taniguchia, Jun Nakayamab,c, Masaaki Shioharae, Eiko Hidakac, Tsutomu Katsuyamac, Sumio Murased, and Junji Sagaraa
a Department of Molecular Oncology and Angiology, Research Center on Aging and Adaptation
b Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine
c Department of Laboratory Medicine, Shinshu University School of Medicine, Shinshu, Japan
d Division of Medical Informatics, Shinshu University Hospital
e Department of Pediatrics, Matsumoto, Nagano, Japan

Correspondence to: Junji Sagara, Dept. of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinshu U. School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Nagano, Japan.


  Summary
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Materials and Methods
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Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a pyrin N-terminal homology domain (PYD)- and caspase recruitment domain (CARD)-containing a proapoptotic molecule. This molecule has also been identified as a target of methylation-induced silencing (TMS)-1. We cloned the ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody. In this study, we determined the binding site of the anti-ASC monoclonal antibody on ASC and analyzed the expression of ASC in normal human tissues. ASC expression was observed in anterior horn cells of the spinal cord, trophoblasts of the placental villi, tubule epithelium of the kidney, seminiferous tubules and Leydig cells of the testis, hepatocytes and interlobular bile ducts of the liver, squamous epithelial cells of the tonsil and skin, hair follicle, sebaceous and eccrine glands of the skin, and peripheral blood leukocytes. In the colon, ASC was detected in mature epithelial cells facing the luminal side rather than immature cells located deeper in the crypts. These observations indicate that high levels of ASC are abundantly expressed in epithelial cells and leukocytes, which are involved in host defense against external pathogens and in well-differentiated cells, the proliferation of which is regulated.

(J Histochem Cytochem 49:1269–1275, 2001)

Key Words: ASC, TMS-1, tissue distribution, PYD, CARD, apoptosis, differentiation


  Introduction
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APOPTOSIS plays important roles in regulating the growth and development of organisms and also mediates normal and neoplastic tissue growth by the removal of excess cells (Kerr et al. 1972 ; Arends and Wyllie 1991 ). Apoptosis is implemented by the death machinery linked to signaling pathways in which death adaptor domains act together in the effector-proximal part of apoptotic signaling components (Nunez et al. 1998 ; Hofmann 1999 ). The caspase recruitment domain (CARD) was proposed as the third example of a heterodimerization domain involved in apoptosis signaling pathways in addition to the death domain (DD) and the death effector domain (DED) (Chinnaiyan et al. 1995 ; Feinstein et al. 1995 ; Hofmann et al. 1997 ).

ASC, composed of an N-terminal pyrin N-terminal homology domain (PYD) and a C-terminal CARD, oligomerizes and enhances anti-cancer drug-induced apoptosis (Masumoto et al. 1999 ). ASC was also identified as target of methylation-induced silencing (TMS)-1, because methylation of CpG islands causes loss of TMS-1 expression (Conway et al. 2000 ). The oligomerization of TMS-1 is accompanied by activation of caspase-9 (McConnell and Vertino 2000 ).

Although we initially cloned ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody, which domain of ASC is recognized by the antibody has not been determined. In this study we determined the site on ASC recognized by the anti-ASC monoclonal antibody and analyzed the cellular distributions of ASC in various tissues to obtain information about the cells in which ASC functions. Here we report that ASC is differentially expressed in human tissues in a manner dependent on both maturation and cell type.


  Materials and Methods
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Construction of Expression Plasmids
The entire open reading frame of ASC was inserted into the EcoRI and SalI sites of pEGFP-C2 (CLONTECH; Palo Alto, CA) to produce pEGFP-ASC. Deletion mutants of pEGFP-ASC-{Delta}1-37, pEGFP-ASC-{Delta}1-58, pEGFP-ASC-{Delta}1-100 (CARD), and pEGFP-ASC-{Delta}101-195 (PYD) were constructed by polymerase chain reaction (PCR) using including a {lambda}gt11 clone of the entire open reading frame of ASC (Masumoto et al. 1999 ) as a template after insertion into the EcoRI and SalI sites of pEGFP-C2.

Transfection, Expression of GFP-fused Proteins and Determination of the Binding Site of the Anti-ASC Monoclonal Antibody
Approximately 1 x 106 COS-7 cells were transfected with expression plasmids using LipofectAMINE-PLUS reagent (Life Technologies; Rockville, MD) according to the manufacturer's instructions. Localizations of GFP-ASC-WT, GFP-ASC-{Delta}1-37, GFP-ASC-{Delta}1-58, GFP-ASC-{Delta}1-100 (CARD), and GFP-ASC-{Delta}101-195 (PYD) in transfected COS-7 cells were analyzed by immunofluorescence microscopy (Zeiss; Oberkochen, Germany). Transfected COS-7 cells were lysed with SDS sample buffer (Laemmli 1970 ), and detected by Western blotting using the anti-ASC monoclonal antibody or an anti-GFP monoclonal antibody.

In Situ Hybridization of ASC Transcripts and Immunohistochemistry of ASC
To detect ASC transcripts in normal human placenta, in situ hybridization was carried out using a non-radioactively labeled RNA probe. Paraffin-embedded blocks of normal human placenta fixed for 48 hr in 20% buffered formalin (pH 7.4) were selected from the pathology files of the Central Clinical Laboratories, (Shinshu University Hospital, Matsumoto, Japan). An ASC-specific nucleotide sequence (nucleotides 101–250) was amplified by PCR using upstream primer (5'-GCTCTAGACGACGCCATCCTGGATGCGC-3') and 3'-primers (5'-GGGGTACCTTGTCGGTGAGGTCCAAGGC-3'), where the Xba I and Kpn I sites are underlined. This amplified DNA fragment was cloned into the Xba I and Kpn I sites of pGEM-3Zf (+) (Promega; Madison, WI), and the resultant vector was used for construction of the RNA probe. A digoxigenin-labeled antisense RNA probe was obtained using Xba I-digested template and T7 RNA polymerase with DIG RNA labeling mixture (Roche Molecular Biochemicals; Mannheim, Germany). Similarly, a sense probe was prepared for negative control experiments using a Kpn I-digested template and SP6 RNA polymerase with the DIG RNA labeling mixture, and ISH was performed as described previously (Kawakami and Nakayama 1997 ). In parallel, immunohistochemical analysis of ASC was carried out using the anti-ASC monoclonal antibody developed in our previous study (Masumoto et al. 1999 ). Paraffin-embedded blocks of various human tissues, including the placenta and spinal cord, were selected from the pathology files of the Central Clinical Laboratories and IHC detection was performed using a universal DAKO LSAB kit (DAKO; Carpinteria, CA), followed by hematoxylin or Giemsa counterstaining. A control experiment was performed by omitting the primary antibody from the staining procedure, and no specific staining was found.

Isolation of Polymorphonuclear Leukocytes, Monocytes, T-lymphocytes, and B-lymphocytes from Human Peripheral Blood
Human peripheral leukocytes obtained from ourselves and our colleagues with their informed consent were isolated by Ficoll–Paque (Amersham Pharmacia Biotech; Uppsala, Sweden) centrifugation of heparinized peripheral blood under conditions described previously (Berkow et al. 1983 ). Red blood cells were lysed with 33 mM NaCl solution added to the same volume of 270 mM NaCl solution, followed by separation of leukocytes. The leukocytes were suspended in RPMI 1640 medium containing 10% (v/v) FBS and incubated at 37C. FITC-conjugated CD3, PE-conjugated CD20, and FITC-conjugated CD14 were purchased from Beckton–Dickinson (Mountain View, CA). Cells were purified with a fluorescence-activated cell sorter. Peripheral blood mononuclear cells were collected in plastic tubes and incubated with appropriately diluted FITC- or PE-conjugated monoclonal antibodies. The cells were washed twice, then analyzed and sorted with a FACS Vantage SE flow cytometer (Beckton–Dickinson). Viable cells were gated according to their forward light-scatter characteristics (FSCs) and side-scatter characteristics (SSCs).


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Determination of the Epitope for the Anti-ASC Monoclonal Antibody
Several deletion mutants of ASC or intact ASC fused with GFP (Fig 1A) were overexpressed in COS-7 cells, and the GFP-fusion proteins were detected by Western blotting using an anti-ASC MAb or an anti-GFP MAb (Fig 1B). Then, both GFP-ASC-WT and GFP-ASC-{Delta}101-195 (PYD) were detected using the anti-ASC MAb, whereas GFP-ASC-{Delta}1-37, GFP-ASC-{Delta}1-58, and GFP-ASC-{Delta}1-100 (CARD) were not detected (Fig 1B, left panel). Expression of GFP constructs was also confirmed by Western blotting using the anti-GFP MAb (Fig 1B, right panel).



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Figure 1. Determination of the sites on ASC reactive with the anti-ASC monoclonal antibody. (A) Schematic representations of wild-type and deletion mutants fused with GFP. (B) 1 x 106 COS-7 cells were transiently transfected with pEGFP-WT (2.4 µg), pEGFP-{Delta}1-37 (2.4 µg), pEGFP-{Delta}1-58 (2.4 µg), pEGFP-{Delta}1-100 (CARD) (2.4 µg), pEGFP-{Delta}101-195 (PYD) (2.4 µg), or pEGFP-C2 (vector) (2.4 µg). After 24 hr, cells were lysed in SDS sample buffer and analyzed by Western blotting using an anti-ASC MAb (left) or anti-GFP MAb (right). Both GFP-ASC-WT and GFP-{Delta}101-195 (PYD) were reacted with anti-ASC MAb, whereas GFP-{Delta}1-37, GFP-{Delta}1-58, and GFP-{Delta}1-100 (CARD) were not (left). The expression of GFP fusion proteins was confirmed by Western blotting (right). The sizes of protein standards in kilodaltons are shown at left. Asterisks indicate truncated products from GFP fusion proteins.

Figure 2. Subcellular localization of GFP-fused deletion mutants of ASC. COS-7 cells were transiently transfected with pEGFP-WT, pEGFP-ASC-{Delta}1-100 (CARD), pEGFP-ASC-{Delta}101-195 (PYD), or pEGFP-C2 (vector control). After 24 hr, green fluorescence signals in the COS-7 cells were detected by immunofluorescence microscopy. (Aa) COS-7 cells were transfected with pEGFP-WT. A speck-like signal (green) was seen in living COS-7 cells. (Ab) Blue nuclei stained with DAPI in the same field. (Ac) Overlay of two channels in the same field. (Ba) COS-7 cells were transfected with pEGFP-ASC-{Delta}1-100 (CARD). Filament-like aggregates of ASC-CARD were seen in COS-7 cells. (Bb) Blue nuclei stained with DAPI in the same field. (Bc) Overlay of two channels in the same field. (Ca) COS-7 cells were transfected with pEGFP-ASC-{Delta}101-195 (PYD). Filament-like aggregates of ASC-PYD were seen in COS-7 cells. (Cb) Blue nuclei stained with DAPI in the same field. (Cc) Overlay of two channels in the same field. (Da) COS-7 cells were transfected with pEGFP-C2 (vector control). Diffuse cytoplasmic signal was observed. (Db) Blue nuclei stained with DAPI in the same field. (Dc) Overlay of two channels in the same field. Bar = 5 µm.

Figure 3. Identification of ASC expression in the human placenta by two different methods: ISH analysis and histochemistry. (A) Schematic representation of ISH probe of ASC (GenBank accession number AB023416). (Ba) ISH analysis in the normal placenta with antisense probe. (Bb) The ASC gene was shown to be expressed in syncytiotrophoblasts (arrowhead) and cytotrophoblasts (arrow) in high-power views. (Bc) No signals were detected in the control with the sense probe. (Ca) Histochemical analysis of the normal placenta with anti-ASC MAb (Cb) ASC staining was also detected in syncytiotrophoblasts (arrowhead) and cytotrophoblasts (arrow) in high-power views. (Cc) No staining was detected in the negative control. Bars: Ba,Bc,Ca,Cc = 40 µm; Bb,Cb = 20 µm.

Figure 4. Expression of ASC in various cells of normal human tissues. (Aa) Expression of ASC in the spinal cord. (Ab) ASC staining was seen in anterior horn cells (arrow) in high-power views. (Ac) No staining was detected in the negative control. (Ba) Expression of ASC in the kidney. ASC staining was not seen in glomeruli (arrowhead). (Bb) ASC staining was seen in proximal tubules in high power views (arrow). ASC was distributed in the periphery of tubule epithelial cells with chromatin condensation (double arrowheads). (Bc) No staining was detected in the negative control. (Ca) Expression of ASC in the testis. (Cb) ASC staining was detected in seminiferous tubules (arrow) and Leydig cells (arrowhead) in high-power views. (Cc) No staining was detected in the negative control. (Da) Expression of ASC in the liver. (Db) ASC staining was detected in interlobular bile duct (arrow) in high-power views. (Dc) ASC staining was also detected in hepatocytes in high power views. (Dd) No staining was detected in the negative control. Bars: Aa,Ac,Ba,Bc,Ca,Cc,Da,Dd = 40 µm; Ab,Bb,Cb,Db,Dc = 10 µm. The results are summarized in Table 1.

Subcellular Localizations of the Deletion Mutants of ASC Fused to GFP Were Analyzed by Fluorescence Microscopy
The subcellular localizations of deletion mutants of ASC presented in Fig 1A were examined. GFP-ASC-WT appeared as perinuclear speck-like aggregates in the transfected COS-7 cells (Fig 2Aa–2Ac). GFP-ASC-{Delta}1-37 (data not shown), GFP-ASC-{Delta}1-58 (data not shown), GFP-ASC-{Delta}1-100 (CARD) (Fig 2Ba–2Bc), and GFP-ASC-{Delta}101-195 (PYD) (Fig 2Ca–2Cc) appeared as filament-like aggregates in the transfected COS-7 cells. GFP, used as a control, was diffusely localized in the cytoplasm in transfected COS-7 cells (Fig 2Da–2Dc).

ASC Expression in the Human Placenta Was Identified by ISH Analysis and IHC
Because we had no data about the distribution of ASC in individual cells, we chose the placenta because it includes a mixture of various types of cells. We analyzed ASC expression in the placenta by two independent experimental methods. We examined whether the data regarding ASC expression obtained by histochemical analysis using the anti-ASC MAb were the same as those for ASC gene expression determined by ISH analysis with an ASC-specific DNA probe (Fig 3A). By ISH, definite signals for ASC mRNA were demonstrated in syncytiotrophoblasts and cytotrophoblasts (Fig 3Ba–3Bc), in which ASC protein was actually detected by IHC with the anti-ASC MAb (Fig 3Ca–3Cc). These results established that the placental trophoblasts lining chorionic villi synthesize ASC and that the anti-ASC MAb specifically recognizes this particular protein.

ASC Expression in Normal Human Tissues Was Demonstrated by IHC
We examined the tissue distribution of ASC in normal human tissues using the anti-ASC MAb in paraffin-embedded sections from a variety of tissues. ASC was detected in a variety of cells, including those of the placenta, anterior horn cells of the spinal cord (Fig 4Aa–4Ac), renal tubules of the kidney (Fig 4Ba–4Bc), seminiferous tubules and Leydig cells of the testis (Fig 4Ca–4Cc), hepatocytes and interlobular bile ducts of the liver (Fig 4Da–4Dd), squamous epithelium of the tonsil (Fig 5Aa–5Ac) and skin (Fig 5Ba–5Bc), hair follicles, sebaceous and eccrine glands of the skin (Fig 5Ba–5Bc), epithelial cells in the colon (Fig 6Aa–6Ac), and peripheral blood leukocytes (data not shown). No significant ASC expression was detected in the ciliated epithelium of the trachea (data not shown), glomeruli of the kidney (Fig 4Ba), cardiac muscle, alveolar epithelium of the lung, or lymphocytes (the lymphatic follicle of the tonsil is shown in Fig 5Ab). The results are summarized in Table 1. We also observed that ASC appeared as speck-like aggregates in peripheral blood leukocytes separated and incubated for 24 hr (data not shown) similar to those observed in apoptotic cells (Masumoto et al. 1999 ; McConnell and Vertino 2000 ). Although the ASC aggregates might also be present in apoptotic tissues, these speck-like aggregates were difficult to detect in the thin tissue sections. However, cytoplasmic ASC staining was redistributed to the cellular periphery in tubular epithelial cells with chromatin condensation (Fig 4Bb, double arrowheads).



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Figure 5. Expression of ASC in the tonsil and skin. (Aa) ASC staining was seen in the squamous epithelium in the oral cavity (arrow). (Ab) ASC was also expressed in the squamous epithelium (arrow) but not in the follicular lymphocytes (*). (Ac) No staining was detected in the negative control. (Ba) Expression of ASC in the skin. ASC was detected in the squamous epithelium (double arrowheads), hair follicles (double arrows), sebaceous (arrowhead), and eccrine glands (arrow). (Bb) ASC staining was detected in the squamous epithelium (arrowheads) in high-power views. (Bc) No staining was detected in the negative control. Bars: Aa–Ac = 20 µm; Ba,Bc = 100 µm; Bb = 10 µm.

Figure 6. Expression of ASC in the colon. (Aa) ASC was expressed in the colon. (Ab) ASC staining was seen in mature luminal epithelial cells (arrowhead) but no expression was detected in immature epithelial cells located deeper in the crypts (arrow) in high-power views. (Ac) No staining was detected in the negative control. Bars: Aa = 100 µm; Ab,Ac = 50 µm.

Figure 7. Protein expression of ASC in normal human tissues and separated peripheral leukocytes analyzed by Western blotting. (A) Forty µg of protein from human normal tissues was analyzed with human anti-ASC MAb. The ASC band is indicated by an arrowhead at right. (B) 1 x 105 cells of protein was analyzed with human anti-ASC MAb. ASC was expressed primarily in CD14-positive monocytes in peripheral blood leukocytes. The ASC band is indicated by an arrowhead at right.


 
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Table 1. Summary of the cellular distributions of ASC in various tissues

ASC Expression in Normal Human Tissues Was Analyzed by Western Blotting
We also examined the expression of ASC using the anti-ASC MAb in normal human tissues from frozen samples and in CD14-positive monocytes, CD3-positive T-lymphocytes, CD20-positive B-lymphocytes, and polymorphonuclear leukocytes (PMNs) separated by cell sorting. ASC expression was detected in the spleen, small intestine, and colon. A trace amount of ASC expression was detected in the kidney (Fig 7A). In peripheral blood, a high level of ASC expression was detected in CD14-positive monocytes, and low levels were detected in PMNs and CD3-positive T-lymphocytes (Fig 7B).


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Anti-ASC MAb Bound to PYD of ASC
The anti-ASC MAb utilized in this study was used for the initial immunoscreening to identify ASC (Masumoto et al. 1999 ). However, its binding site on ASC had not been determined. Fig 1 shows that the anti-ASC MAb reacts with a region around the N-terminal 37 residue of ASC. In addition, two ASC cDNA clones of 425 and 725 base pairs, which were obtained by initial immunoscreening with the anti-ASC MAb, encoded the entire PYD domain (Masumoto et al. 1999 ). These results indicated that the anti-ASC MAb reacted with the PYD of ASC. Furthermore, the anti-ASC MAb did not crossreact with the murine orthologue of ASC (mASC) (Masumoto et al. 2001a ). Because the N-terminal 15 residues of mASC are entirely conserved, the anti-ASC MAb is believed to react with a region including residues 16–30 of the PYD of ASC.

Is ASC a Possible Key Adaptor Molecule Between PYD-containing Proteins and CARD-containing Proteins?
The speck-like aggregation of GFP-ASC-WT was different from the filament-like aggregation of GFP-ASC-{Delta}1-100 (CARD) or GFP-ASC-{Delta}101-195 (PYD). This was believed to be due to an intramolecular heterophilic interaction between PYD and CARD, which is involved in the oligomerization of ASC (Masumoto et al. 2001b ). The PYD domain has been referred to as the PYRIN, DAPIN, PAAD, and pyrin domain, and a number of PYD-containing proteins were reported while this article was in preparation (Bertin and DiStefano 2000 ; Martinon et al. 2001 ; Pawlowski et al. 2001 ; Staub et al. 2001 ). The recently identified proapoptic molecule DEFCAP/NAC/NALP1/CARD7 (Bertin and DiStefano 2000 ; Chu et al. 2001 ; Hlaing et al. 2001 ; Martinon et al. 2001 ), a member of the mammalian Ced-4 protein family that includes Apaf-1 and Nod1, which contain an N-terminal PYD domain and leucine-rich repeats (LRR), binds to ASC, referred to as pycard by Martinon et al. 2001 , via the PYD. The PYD domain was reported to be present in the N-terminus of the zebrafish caspases Caspy and Caspy2 (Inohara and Nunez 2000 ). The PYD domains of Caspy and Caspy2 correspond to CARD or DED, which are involved in protein–protein interactions that result in effector proximity (Hofmann 1999 ). Therefore, the ASC may be a key adaptor molecule that plays important roles in interaction with other PYD-containing molecules through its PYD and CARD domains.

Protein Expression of ASC Was Detected in the Epithelial Surface and in Some Differentiated Functional Cells for Which Proliferation is Regulated
The expression patterns of ASC associated with differentiation in squamous epithelium in the skin (Fig 5Ba–5Bc), tonsil (Fig 5Aa–5Ac), and intestinal mucosal epithelium (Fig 6Aa–6Ac), were very similar (Chu et al. 2001 ), consistent with the binding of ASC to DEFCAP/NAC/NALP1/CARD7 (Martinon et al. 2001 ). On the other hand, significant ASC expression was not detected in the ciliated epithelium of the trachea, glomeruli of the kidney, cardiac muscle, alveolar epithelium of the lung, or lymphocytes (Table 1), although these cell types are also believed to be well-differentiated. DEFCAP/NAC/NALP1/CARD7 is a multiple leucine-rich repeat (LRR)-containing mammalian Ced4 family protein that is believed to interact with some pathogens. Other members of the mammalian Ced4 protein family, including human Nod1/CARD4 and Nod2, confer responsiveness to bacterial lipopolysaccharides (Inohara et al. 2001 ; Ogura et al. 2001 ). By interaction with DEFCAP/NAC/NALP1/CARD7 or other mammalian Ced4 members, ASC may activate downstream effector proteins involved in apoptosis and host defense against external pathogens.

We previously reported high levels of ASC mRNA expression in the spleen, which contains large numbers of lymphocytes (Masumoto et al. 1999 ), but no significant ASC expression was detected in individual lymphocytes. Our fractionation indicated that ASC was primarily expressed in CD14-positive monocytes in peripheral blood leukocytes (Fig 7). Therefore, we speculated that this signal in the spleen might have been due to large numbers of splenic monocytes/follicular dendritic cells and accumulation of trace levels of ASC expressed in individual CD3-positive T-lymphocytes. It is also noteworthy that ASC was expressed in epithelial cells located in the upper region of the colon mucosa rather than those in deeper regions (Fig 6Aa–6Ac). Cell number in the gastrointestinal tract is regulated by apoptosis occurring in the upper region of the mucosa (Hall et al. 1994 ). This result suggested that ASC might be involved in maturation of colon epithelial cells and host defense against surface pathogens.

Recently, ASC was independently identified as TMS-1 and was shown to confer lack of ASC expression through methylation-mediated silencing, a survival advantage (Conway et al. 2000 ; McConnell and Vertino 2000 ). Therefore, ASC expression may be involved in suppression of cell growth to regulate normal differentiation and responses to some pathogens. Although the precise functions of ASC in the tissues are not yet clear, ASC was differentially expressed in human tissues in a manner dependent on both maturation and cell type.


  Acknowledgments

Supported by a Grant-in-Aid 12670109 from the Ministry of Education, Science and Culture, Japan.

We thank Drs Hiroshi Zenda and Shin Ohta (Department of Pharmacy, Shinshu University Hospital) for encouragement during this work and Masanobu Momose (Central Clinical Laboratory, Shinshu University Hospital) for technical assistance.

Received for publication January 29, 2001; accepted May 16, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Arends MJ, Wyllie AH (1991) Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32:223-254[Medline]

Berkow RL, Tzeng DY, Williams LV, Baehner RL (1983) The comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll-Hypaque density centrifugation. I. Resting volume, stimulus-induced superoxide production, and primary and specific granule release. J Lab Clin Med 102:732-742[Medline]

Bertin J, DiStefano PS (2000) The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ 7:1273-1274[Medline]

Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81:505-512[Medline]

Chu ZL, Pio F, Xie Z, Welsh K, Krajewska M, Krajewski S, Godzik A, Reed JC (2001) A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis. J Biol Chem 276:9239-9245[Abstract/Free Full Text]

Conway KE, McConnell BB, Bowring CE, Donald CD, Warren ST, Vertino PM (2000) TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res 60:6236-6242[Abstract/Free Full Text]

Feinstein E, Kimchi A, Wallach D, Boldin M, Varfolomeev E (1995) The death domain: a module shared by proteins with diverse cellular functions. Trends Biochem Sci 20:342-344[Medline]

Hall PA, Coates PJ, Ansari B, Hopwood D (1994) Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107:3569-3577[Abstract/Free Full Text]

Hlaing T, Guo RF, Dilley KA, Loussia JM, Morrish TA, Shi MM, Vincenz C, Ward PA (2001) Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins. J Biol Chem 276:9230-9238[Abstract/Free Full Text]

Hofmann K (1999) The modular nature of apoptotic signaling proteins. Cell Mol Life Sci 55:1113-1128[Medline]

Hofmann K, Bucher P, Tschopp J (1997) The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci 22:155-156[Medline]

Inohara N, Ogura Y, Chen FF, Muto A, Núñez G (2001) Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem 276:2551-2554[Abstract/Free Full Text]

Inohara N, Núñez G (2000) Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ 7:509-510[Medline]

Kawakami M, Nakayama J (1997) Enhanced expression of prostate-specific membrane antigen gene in prostate cancer as revealed by in situ hybridization. Cancer Res 57:2321-2324[Abstract]

Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257[Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]

Martinon F, Hofmann K, Tschopp J (2001) The pyrin domain: a possible member of the death domain-fold family implicated in apoptosis and inflammation. Curr Biol 11:R118-120[Medline]

Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N, Hidaka E, Katsuyama T, Higuchi T, Sagara J (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274:33835-33838[Abstract/Free Full Text]

Masumoto J, Taniguchi S, Nakayama K, Ayukawa K, Sagara J (2001a) Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp Cell Res 262:128-133[Medline]

Masumoto J, Taniguchi S, Sagara J (2001b) Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC. Biochem Biophys Res Commun 280:652-655[Medline]

McConnell BB, Vertino PM (2000) Activation of a caspase-9-mediated apoptotic pathway by subcellular redistribution of the novel caspase recruitment domain protein TMS1. Cancer Res 60:6243-6247[Free Full Text]

Núñez G, Benedict MA, Hu Y, Inohara N (1998) Caspases: the proteases of the apoptotic pathway. Oncogene 17:3237-3245[Medline]

Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G (2001) Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF{kappa}B. J Biol Chem 276:4812-4818[Abstract/Free Full Text]

Pawlowski K, Pio F, Chu Z, Reed JC, Godzik A (2001) PAAD—a new protein domain associated with apoptosis, cancer and autoimmune diseases. Trends Biochem Sci 26:85-87[Medline]

Staub E, Dahl E, Rosenthal A (2001) The DAPIN family: a novel domain links apoptotic and interferon response proteins. Trends Biochem Sci 26:83-85[Medline]