Cancer Research Meeting Calendar Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McConnell, B. B.
Right arrow Articles by Vertino, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McConnell, B. B.
Right arrow Articles by Vertino, P. M.
[Cancer Research 60, 6243-6247, November 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Activation of a Caspase-9-mediated Apoptotic Pathway by Subcellular Redistribution of the Novel Caspase Recruitment Domain Protein TMS11

Beth B. McConnell and Paula M. Vertino2

Department of Radiation Oncology and the Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Genetic and epigenetic alterations affecting proteins involved in apoptosis can contribute to the establishment and progression of cancer. Recently, our laboratory has isolated a novel gene, TMS1, that is aberrantly methylated and silenced in a significant proportion of human breast cancers. TMS1 contains a caspase recruitment domain (CARD), suggesting a role in caspase-mediated cell death. In the present study, we characterize the participation of TMS1 in apoptosis and examine the subcellular localization of the protein. Inducible expression of TMS1 inhibited cellular proliferation and induced DNA fragmentation in a time-dependent manner. These apoptotic events were blocked by the general caspase inhibitor, Z-VAD-fmk. The ability of TMS1 to trigger apoptosis was also suppressed by a dominant negative form of caspase-9 but not by a dominant negative form of caspase-8, indicating that TMS1 functions through activation of caspase-9. Unlike a number of other CARD-containing proteins, TMS1 did not activate nuclear factor {kappa}B-dependent transcription, consistent with a proapoptotic role for TMS1 in death signaling pathways. Timed localization studies revealed that TMS1-induced apoptosis was accompanied by the redistribution of TMS1 from the cytoplasm to perinuclear spherical structures. Whereas the apoptotic activity of TMS1 was blocked by caspase inhibition, the formation of TMS1-containing subcellular structures was not, suggesting that the redistribution of TMS1 precedes caspase activation. Both the proapoptotic activity of TMS1 and aggregate formation were dependent on the CARD. In summary, the data indicate that TMS1-induced apoptosis proceeds through a CARD-dependent aggregation step followed by activation of a caspase-9-mediated pathway.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Apoptosis, or programmed cell death, is a mechanism that exists in multicellular organisms whereby individual cells orchestrate their own deletion in response to cellular damage, infection, or external stimuli (1) . The ability of cells to respond to regulatory signals to undergo apoptosis can be lost in transformed cells, conferring a survival advantage. Apoptosis has been shown to play a major role in limiting the population expansion of tumor cells early in the process of tumor growth after initial transformation events (2) . In addition, survival of metastatic tumor cells appears to be enhanced by lack of normal responses to apoptotic signals (3) . Thus, loss of apoptotic signaling mechanisms contributes to both the initiation and progression of human cancers.

Apoptosis is mediated through the activity of a family of cysteine proteases known as caspases that trigger apoptosis through a hierarchical cascade of cleavage events (4) . Caspases exist in the cell as latent proenzymes until cleaved into their active forms. The apical caspases-8 and -9 are activated through their association with adaptor proteins via homologous protein/protein interaction domains (5) . One recently defined domain, the CARD,3 has been shown to mediate the interaction between the adaptor protein Apaf-1 and the proform of caspase-9 in response to release of cytochrome c from the mitochondria (6) . CARD regions have also been identified in the prodomains of caspase-1, -2, -4, -11, and -12 as well as in the adaptor and regulatory proteins RAIDD, RICK, c-IAP1, and c-IAP2 (7) . All CARD proteins identified thus far have been shown to participate in apoptosis.

There is growing evidence that altered expression of CARD-containing regulatory molecules may play an important role in carcinogenesis. Two CARD-encoding genes, API2 and BCL10, have been identified at translocation breakpoints in mucosal-associated lymphoid tissue lymphomas (8, 9, 10) . In addition, our laboratory has identified a novel CARD-encoding gene, TMS1, that is silenced by aberrant methylation in human breast cancers (11) . Here, we characterize the role of TMS1 in programmed cell death. We find that TMS1 promotes apoptosis, and its activity is caspase dependent. In particular, TMS1-induced cell death requires the activation of caspase-9. We also find that TMS1-induced apoptosis is accompanied by the redistribution of TMS1 from the cytoplasm to perinuclear structures and that formation of these structures occurs upstream of caspase activation. Given the proapoptotic role of TMS1, methylation-induced silencing of the TMS1 gene in cancer cells may contribute to escape from apoptosis, a powerful selective advantage in carcinogenesis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Plasmids.
pcDNA-TMS1 and pcDNA-mycTMS1 have been described in the accompanying article (11) . The TMS1 COOH-terminal truncation mutant (mycTMS1{Delta}100–195) was created from pcDNA-mycTMS1 by Klenow fill-in of an internal BamHI site, resulting in a frameshift at amino acid 100 followed by an in-frame stop codon. The DN caspase-8 (12) and DN caspase-9 (6) constructs were gifts from K. Bhalla (H. Lee Moffitt Cancer Center, Tampa, FL). The NF-{kappa}B CAT (pJECAT2.6) and mutant NF-{kappa}B CAT (p2.6m{kappa}B1) constructs were gifts from J. Boss (Emory University, Atlanta, GA) (13) .

Creation of Cells with Inducible TMS1 Expression.
The Ecdysone-Inducible Mammalian Expression System (Invitrogen, Carlsbad, CA) was used to create clones of 293 cells that inducibly express mycTMS1. The mycTMS1 cDNA was cloned into the HindIII/XhoI sites of the pIND expression vector (pIND-mycTMS1). EcR-293 cells (Invitrogen) containing the pVgRXR vector were transfected with pIND-mycTMS1 or pIND using LipofectAMINE reagent (Life Technologies, Inc., Grand Island, NY). Cells were maintained in selection medium containing 600 µg/ml G418 for 3 weeks to isolate stably transfected colonies. Clonal populations transfected with pIND-mycTMS1 were then tested for inducible expression by Western blot analysis after the addition of 5 µM ponA.

Cell Culture and Transfection.
Human embryonic kidney 293 cells and EcR-293 derivatives (MTMS22 and PIND1) were cultured in DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. MTMS22 and PIND1 cells were maintained in the presence of 400 µg/ml zeocin and 600 µg/ml G418. Transfections of 293 cells were carried out in 24-well dishes with 1 x 105 cells and 0.5 µg of total DNA per well using the calcium phosphate method. As a transfection marker, a ß-gal expression vector (ß-gal CMV) was included at a 1:4 ratio with the indicated cDNA constructs. In cotransfections with DN caspase-8 or DN caspase-9, 0.2 µg of pcDNA-TMS1 was transfected with 0.2 µg of pcDNA3.1 or DN caspase-8 or -9 along with 0.1 µg of ß-gal CMV as a transfection control.

Morphological Apoptosis Assay.
The 293 cells were fixed on coverslips and stained for ß-gal activity 48 h after transfection. Nuclei were stained using Hoechst 33258 dye (Sigma, St. Louis, MO). Transfected (blue) cells were examined for morphological changes indicative of adherent cells undergoing apoptosis including cell rounding and reduction in size, nuclear fragmentation, and membrane blebbing.

DNA Fragmentation.
Where indicated, MTMS22 cells were treated with 5 µM ponA (Invitrogen) or 40 µM Z-VAD-fmk (Enzyme System Products, Livermore, CA). DNA was collected from 2 x 106 cells as described previously (14) . DNA (5 µg) from each sample was visualized by separation on a 2% agarose gel containing ethidium bromide.

CAT Assay for NF-{kappa}B Activation.
The 293 cells plated at 60% confluence in 6-well dishes were transfected with 0.2 µg of the NF-{kappa}B CAT reporter construct (pJECAT2.6) and increasing amounts of pcDNA-mycTMS1 by the calcium phosphate method. ß-Gal CMV (0.2 µg) was included as a transfection control, and the total amount of DNA transfected was kept constant at 2 µg with pcDNA3.1. Cell lysates were collected 36 h after transfection in ß-gal lysis buffer and assayed for ß-gal activity using the ß-Gal Enzyme Assay System (Promega, Madison, WI) per the manufacturer’s instructions. CAT assays were performed basically as described previously (15) . In brief, cell lysates were incubated with [14C]chloramphenicol and n-butyryl-CoA. 14C-labeled acetylated chloramphenicol was then separated from the nonacetylated form by organic phase extraction, and radioactive counts were determined using a scintillation counter.

Fluorescence Microscopy.
Cells were fixed in 4% formaldehyde, permeabilized in 0.2% Triton X-100 in PBS, and blocked with 3% BSA/0.02% Triton X-100 in PBS. Coverslips were incubated with myc antibody (9E10) (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution, washed four times in PBS/0.02% Triton X-100, and incubated with a secondary FITC antibody at a 1:2000 dilution. After three more washes, cells were stained with Hoechst 33258 dye to visualize nuclei, washed twice in PBS, and mounted on slides. Cells were viewed at x400 or x1000 using the Olympus BX60 microscope. Digital images were captured using IP Lab Spectrum v.3.1 software (Scanalytics, Inc., Fairfax, VA).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
An ecdysone-regulated expression system was used to create a 293 cell line that inducibly expresses TMS1. A clonal population (called MTMS22) that exhibited no detectable background expression of TMS1 and high inducible expression in the presence of the ecdysone analogue ponA was isolated and used for the following studies. In addition, a clonal population of cells stably transfected with the empty expression vector was isolated as a control (PIND1). On addition of ponA to the MTMS22 cells, mycTMS1 was detectable by Western blotting as early as 6 h after treatment and continued to accumulate in the cells over 48 h (Fig. 1A)Citation . To determine whether TMS1 had an effect on cellular proliferation, MTMS22 cells were counted over a 96-h period in the presence or absence of ponA. By 48 h after induction of TMS1, cells had begun to round up and lift off the plates (data not shown), and cell growth was significantly inhibited (Fig. 1B)Citation . By 96 h, ponA-treated cells were declining in number, confirming cell death. As a biochemical marker for apoptosis, MTMS22 cells were examined for DNA fragmentation in the presence or absence of TMS1 expression. On induced expression of TMS1, DNA laddering was observed as early as 16 h and was most evident at 48 h (Fig. 1C)Citation . To address whether the death-inducing effects of TMS1 were caspase dependent, these experiments were also carried out in the presence of the general caspase inhibitor, Z-VAD-fmk. The addition of Z-VAD blocked both TMS1-induced cell death (Fig. 1B)Citation and DNA fragmentation (Fig. 1C)Citation . Neither ponA or Z-VAD had any effect on the growth of PIND1 control cells (data not shown). These data indicate that TMS1 expression induces apoptosis, and this activity is dependent on caspase activation.



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Induction of caspase-dependent apoptosis by TMS1. A, an ecdysone-inducible expression system was used to express myc-tagged TMS1. MTMS22 cells were treated with ponA for the indicated times, and TMS1 expression was examined by immunoblotting with a monoclonal myc (9E10) antibody. B, growth of MTMS22 cells in the presence or absence of 5 µM ponA with or without 40 µM Z-VAD. Data represent the mean ± SD of triplicate determinations from a representative growth experiment. C, MTMS22 cells were treated with ponA and/or Z-VAD for the indicated times, and DNA fragmentation was visualized by ethidium bromide/agarose gel electrophoresis.

 
To identify candidate caspase pathways in which TMS1 functions, we tested the ability of DN forms of the initiator caspases, caspase-8 and -9, to block TMS1-induced apoptosis. The 293 cells were transiently transfected with TMS1 alone or with TMS1 plus DN caspase-8 or DN caspase-9 and examined for morphological changes associated with apoptosis. Ectopic expression of TMS1 resulted in a 6-fold increase in cell death (Fig. 2A)Citation . The apoptotic activity of TMS1 was not affected by DN caspase-8; in contrast, DN caspase-9 significantly inhibited TMS1-induced apoptosis. The ability of DN caspase-9 to block cell death induced by TMS1 was similar to the effect seen with the general caspase inhibitor, Z-VAD. These data suggest that the proapoptotic activity of TMS1 is mediated predominantly through the activation of caspase-9.



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. TMS1 functions through a caspase-9-mediated pathway and is not involved in NF-{kappa}B signaling pathways. A, proapoptotic activity of TMS1 is dependent on caspase-9. The 293 cells were transiently transfected with pcDNA-TMS1, with pcDNA-TMS1 plus DN caspase-8 or -9, or with pcDNA-TMS1 in the presence of 40 µM Z-VAD. Forty-eight h after transfection, the percentage of transfected (ß-gal-positive) cells exhibiting morphological features of apoptosis was determined. At least 200 transfected cells were counted per transfection. Data represent the mean ± SD of three separate experiments. B, TMS1 has no effect on NF-{kappa}B activation. The 293 cells were transfected with a NF-{kappa}B CAT reporter construct (pJECAT2.6) and the indicated amounts of pcDNA-mycTMS1. Cells were lysed 36 h later and assayed for CAT activity as described in "Materials and Methods." A CAT reporter construct containing a mutated NF-{kappa}B site, (p2.6m{kappa}B1) was used as a negative control. TNF-{alpha} (20 µM) was added as a positive control. Inset, lysates used for CAT assays were subjected to immunoblot analysis for TMS1.

 
In addition to the ability to trigger or enhance apoptosis, several CARD-containing proteins have been shown to activate the transcription factor NF-{kappa}B. To test whether TMS1 was able to induce NF-{kappa}B-activated transcription, 293 cells were cotransfected with a NF-{kappa}B-responsive CAT reporter construct along with increasing amounts of TMS1. TMS1 expression had no effect on NF-{kappa}B-dependent transcriptional activation (Fig. 2B)Citation . In contrast, the addition of TNF-{alpha} induced a 15-fold increase in activation. Thus, although TMS1 is able to induce apoptosis, it does not appear to participate in NF-{kappa}B activation pathways.

To determine the subcellular localization of TMS1, MTMS22 cells induced to express TMS1 were examined by immunofluorescence. Sixteen h after induction, TMS1 showed diffuse cytoplasmic staining (Fig. 3A)Citation . However, by 24 h, a fraction of the cells showed a punctate fluorescent pattern, and by 48 h, a majority of the cells contained the punctate staining and lacked diffuse cytoplasmic staining. Under high power, the TMS1 aggregates appeared as hollow, spherical structures made up of many smaller balls (Fig. 3B)Citation . For a majority of the cells, there appeared to be only one structure per cell, located in close proximity to the nucleus. Redistribution of TMS1 from the cytoplasm to the aggregates correlated with partial detachment of the cells from the growth surface, resulting in increased refraction of light when viewed by phase-contrast microscopy (Fig. 3A)Citation . The timing of TMS1 relocalization after induction coincided with TMS1-induced cell death and the appearance of DNA fragmentation (compare Fig. 3ACitation with Fig. 1, B and CCitation ). An intriguing result was observed in cells expressing TMS1 in the presence of Z-VAD (Fig. 3A)Citation . By 48 h after induction, most cells contained the aggregate structures, and the overall staining pattern was indistinguishable from that observed in the absence of Z-VAD. Thus, although Z-VAD was able to block the apoptotic effects of TMS1 (Fig. 1, B and C)Citation , it had no effect on the formation of the TMS1-containing spherical aggregates. Therefore, TMS1 aggregate formation is not a downstream effect of apoptosis but rather appears to be an intermediate event that lies upstream of caspase activation.



View larger version (55K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Subcellular localization of TMS1 in 293 cells. A, MTMS22 cells were induced to express myc-tagged TMS1 by addition of ponA for the indicated times and prepared for visualization of TMS1 by immuofluorescence using a myc (9E10) monoclonal antibody as described in "Materials and Methods." Nuclei were visualized by staining with Hoescht 33258 dye. Cells were viewed at x400. B, 293 cells were transiently transfected with pcDNA-mycTMS1 and processed 48 h later to visualize TMS1 and nuclei by immunofluorescence. Images are magnified at x1000.

 
To test the importance of the CARD region for the function and localization of TMS1, a truncated form of TMS1 lacking the COOH-terminal CARD was compared to full-length TMS1 in its ability to induce apoptosis and to localize to the spherical structures. Transient expression of mycTMS1 induced apoptosis in 293 cells, whereas deletion of the CARD (mycTMS1{Delta}100-195) abolished the proapoptotic activity (Fig. 4A)Citation . Western blot analysis confirmed that both TMS1 proteins were expressed at equal levels (Fig. 4B)Citation . Deletion of the CARD also affected subcellular localization of TMS1. Whereas wild-type TMS1 localized to the aggregates, TMS1 lacking the CARD remained almost exclusively cytoplasmic (Fig. 4C)Citation . Thus, both the proapoptotic activity and the localization of TMS1 to spherical structures are dependent on the CARD.



View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. TMS1-induced apoptosis and localization are dependent on the CARD. A, 293 cells were transiently transfected with pcDNA3.1, pcDNA-mycTMS1, or pcDNA-mycTMS1{Delta}100-195. Percentages of apoptotic cells were determined after 48 h by examining the morphology of ß-gal-positive cells. At least 200 transfected cells were counted per transfection, and results represent the mean ± SD of three separate experiments. B, lysates from transfected 293 cells were examined for expression of full-length or truncated TMS1 (top) and ß-gal (bottom) by immunoblot analysis. C, 293 cells transiently transfected with pcDNA-mycTMS1 or pcDNA-mycTMS1{Delta}100-195 were prepared for visualization of TMS1 by immunofluorescence. At least 500 TMS1-positive cells were scored for subcellular localization of TMS1.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Here we characterize TMS1, a novel CARD-containing protein that, with the exception of the CARD, is structurally unrelated to other known CARD adaptor and regulatory proteins. Ectopic expression of TMS1 alone was able to trigger apoptosis in 293 cells, and cell death correlated with relocalization of TMS1 from the cytoplasm to perinuclear, ball-like structures. Several lines of evidence support the idea that redistribution of TMS1 is an intermediate step in a TMS1-triggered apoptotic pathway. First, the aggregate structures were formed in the absence of caspase activity, implying that TMS1 aggregation is not a consequence of apoptosis but rather an event that occurs upstream of caspase activation. In addition, deletion of the CARD region of TMS1 inhibited aggregate formation and abolished the proapoptotic activity of TMS1, suggesting that aggregation is necessary for TMS1-induced apoptosis. Furthermore, Masumoto et al. (16) reported the redistribution of endogenous TMS1 (which they refer to as ASC) from a soluble, cytoplasmic form to insoluble, subcellular aggregates on treatment of HL-60 cells with retinoic acid or etoposide. Thus, aggregation of TMS1 appears to be a causative event in TMS1-triggered cell death.

A growing number of studies suggest that the relocalization and aggregation of apoptotic signaling proteins is an important step in caspase activation. FADD and caspase-8 have been shown to redistribute into ordered, subcellular filaments in cells transfected with FADD (17 , 18) . Localization of FADD and caspase-8 was dependent on their death effector domains, and disruption of the filaments blocked FADD-induced apoptosis. Similarly, Apaf-1, procaspase-9, and cytochrome c have been shown to shift into large, multimeric complexes (>1.3 x 103 kDa) termed apoptosomes to initiate caspase-9 activation (19) . Based on these and similar studies is the induced proximity model that proposes that clustering of receptors and/or adaptor proteins with procaspases leads to caspase activation (20) . Overexpression of TMS1 results in CARD-mediated clustering of the protein, and the structures that are formed may include other proteins whose association can trigger caspase activation and apoptosis.

Our data showed that the proapoptotic activity of TMS1 was caspase dependent and, in particular, required the activity of caspase-9. Caspase-9 is involved in the activation of apoptosis after release of cytochrome c from the mitochondria, an almost universal triggered by numerous event/apoptotic stimuli, including DNA damage and various chemotherapeutic agents (21 , 22) . Masumoto et al. (16) reported that the introduction of ASC (TMS1) antisense oligonucleotides inhibited etoposide-induced apoptosis in HL-60 cells. These results are consistent with our data indicating that TMS1 functions in a caspase-9-dependent pathway. Thus, TMS1 may act as part of a signaling cascade for initiating activation of caspase-9 in response to certain external stimuli.

Several CARD-containing proteins including RICK/RIP2, CARD4/Nod1, and BCL10/CIPER/CLAP, in addition to playing a role in caspase activation, have been shown to trigger NF-{kappa}B-mediated transcription (8 , 9 , 23, 24, 25, 26, 27, 28) . Activation of NF-{kappa}B by DNA-damaging agents or TNF-{alpha} can act as part of a regulatory feedback loop that operates to prevent cell death, most likely by inducing expression of antiapoptotic genes (29) . Indeed, NF-{kappa}B target gene products including cIAP-1, cIAP-2, TRAF1, and TRAF2 have been shown to protect cells from apoptosis induced by TNF-{alpha} (30) . TMS1 had no effect on the activation of NF-{kappa}B and thus is unlikely to participate in survival pathways mediated by this transcription factor.

Genetic alterations that provide resistance to apoptosis promote tumorigenesis by allowing cancer cells to persist and accumulate further genetic damage (31) . There is accumulating evidence that genetic changes affecting the function or expression of CARD proteins can provide such a survival advantage. The gene encoding the proapoptotic CARD-containing protein BCL10 is subject to translocation in mucosal-associated lymphoid tissue B-cell lymphomas, resulting in a variety of truncation mutations that disrupt the ability of BCL10 to activate apoptosis (8 , 9) . Likewise, the TMS1 locus has been shown previously by our laboratory to be silenced by aberrant DNA methylation in human breast cancers (11) . Given the proapoptotic function of TMS1 reported here, loss of expression of this CARD-containing protein through epigenetic alterations can provide cancer cells with a means to escape apoptosis. Because the effects of TMS1 are caspase-9 dependent, loss of TMS1 expression may disrupt normal apoptotic responses to DNA damage or cellular stress, thus providing resistance to irradiation or chemotherapeutic agents.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Gordon Peters for critical review of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants CA77337 (to P. M. V.) and 1 F32 CA83289-01 (to B. B. M.). P. M. V. is an Avon Scholar in Breast Cancer Genomics. Back

2 To whom requests for reprints should be addressed, at Winship Cancer Institute, Room B5119, 1365-B Clifton Road NE, Atlanta, GA 30322. E-mail: pvertin{at}emory.edu Back

3 The abbreviations used are: CARD, caspase recruitment domain; DN, dominant negative; NF-{kappa}B, nuclear factor {kappa}B; TNF, tumor necrosis factor; ß-gal, ß-galactosidase; ponA, ponasterone A; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; FADD, Fas-associated protein with death domain, Z-VAD, z-VAD-fmk. Back

Received 6/19/00. Accepted 10/ 3/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Cohen G. M. Caspases: the executioners of apoptosis. Biochem. J., 326: 1-16, 1997.
  2. Wyllie A. H. Apoptosis and carcinogenesis. Eur. J. Cell Biol., 73: 189-197, 1997.[Medline]
  3. Thompson C. B. Apoptosis in the pathogenesis and treatment of disease. Science (Washington DC), 267: 1456-1462, 1995.[Abstract/Free Full Text]
  4. Salvesen G. S., Dixit V. M. Caspases: intracellular signaling by proteolysis. Cell, 91: 443-446, 1997.[Medline]
  5. Thornberry N. A., Lazebnik Y. Caspases: enemies within. Science (Washington DC), 281: 1312-1316, 1998.[Abstract/Free Full Text]
  6. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Admad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell, 91: 479-489, 1997.[Medline]
  7. Hofmann K. The modular nature of apoptotic signaling proteins. Cell. Mol. Life Sci., 55: 1113-1128, 1999.[Medline]
  8. Willis T. G., Jadayel D. M., Du M-Q., Peng H., Pery A. R., Abdul-Rauf M., Price H., Karran L., Majekodunmi O., Wlodarska I., Pan L., Crook T., Hamoudi R., Isaacson P. G., Dyer M. J. S. Bcl10 is involved in t(1;14)(p22;p32) of MALT B cell lymphoma and mutated in multiple tumor types. Cell, 96: 35-45, 1999.[Medline]
  9. Zhang Q., Siebert R., Yan M., Hinzmann B., Cui X., Xue L., Rakestraw K. M., Naeve C. W., Beckmann G., Weisenburger D. D., Sanger W. G., Nowotny H., Vesely M., Callet-Bauchu E., Salles G., Dixit V. M., Rosenthal A., Schlegelberger B., Morris S. W. Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32). Nat. Genet., 22: 63-68, 1999.[Medline]
  10. Dierlamm J., Baens M., Wlodarska I., Stefanova-Ouzounova M., Hernandez J. M., Hossfeld D. K., De Wolf-Peeters C., Hagemeijer A., Van den Berghe H., Marynen P. The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue lymphomas. Blood, 93: 3601-3609, 1999.[Abstract/Free Full Text]
  11. Conway K. E., McConnell B. B., Bowring C. E., Donald C. D., Warren S. T., Vertino P. M. 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, 2000.[Abstract/Free Full Text]
  12. Muzio M., Chinnaiyan A. M., Kischkel F. C., O’Rourke K., Shevchenko A., Ni J., Scaffidi C., Bretz J. D., Zhang M., Gentz R. FLICE, a novel FADD-like homologous ICE/CED3 protease, is recruited to the CD95(Fas/APO-1) death-inducing signaling complex. Cell, 85: 817-827, 1996.[Medline]
  13. Ping D., Boekhoudt G. H., Rogers E. M., Boss J. M. Nuclear factor-{kappa}B p65 mediates the assembly and activation of the TNF-responsive element of the murine monocyte chemoattractant-1 gene. J. Immunol., 162: 727-734, 1999.[Abstract/Free Full Text]
  14. Burow M. E., Weldon C. B., Tang Y., Navar G. L., Krajewski S., Reed J. C., Hammond T. G., Clejan S., Beckman B. S. Differences in susceptibility to tumor necrosis factor {alpha}-induced apoptosis among MCF-7 breast cancer cell variants. Cancer Res., 58: 4940-4946, 1998.[Abstract/Free Full Text]
  15. Kingston, R. E., Sheen, J., and Moore, D. Isotopic assays for reporter gene activity. In: F. A. Ausubel, R. Brent, R., R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds.), Current Protocols in Molecular Biology, pp. 9.7.6–9.7.7. New York: John Wiley & Sons Inc., 1995.
  16. Masumoto J., Taniguchi S. I., Ayukawa K., Sarvotham H., Kishino T., Niikawa N., Hidaka E., Katsuyama T., Higuchi T., Sagara J. ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J. Biol. Chem., 274: 33835-33838, 1999.[Abstract/Free Full Text]
  17. Siegel R. M., Martin D. A., Zheng L., Ng S. Y., Bertin J., Cohen J., Lenardo M. J. Death-effector filaments: novel cytoplasmic structures that recruit caspases and trigger apoptosis. J. Cell Biol., 141: 1243-1253, 1998.[Abstract/Free Full Text]
  18. Perez D., White E. E1B 19K inhibits fas-mediated apoptosis through FADD-dependent sequestration of FLICE. J. Cell Biol., 141: 1255-1266, 1998.[Abstract/Free Full Text]
  19. Zou H., Li Y., Liu X., Wang X. An Apaf-1-cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem., 274: 11549-11556, 1999.[Abstract/Free Full Text]
  20. Salvesen G. S., Dixit V. M. Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA, 96: 10964-10967, 1999.[Abstract/Free Full Text]
  21. Kuida K., Haydar T. F., Kuan C-Y., Gu Y., Yaya C., Karasuyama H., Su M. S-S., Rakic P., Flavell R. A. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell, 94: 325-337, 1998.[Medline]
  22. Hakem R., Hakem A., Duncan G. S., Henderson J. T., Woo M., Soengas M. S., Elia A., de la Pompa J. L., Kagi D., Khoo W., Potter J., Yoshida R., Kaufman S. A., Lowe S. W., Penninger J. M., Mak T. W. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell, 94: 339-352, 1998.[Medline]
  23. Inohara N., del Peso L., Koseki T., Chen S., Nunez G. RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis. J. Biol. Chem., 273: 12296-12300, 1998.[Abstract/Free Full Text]
  24. McCarthy J. V., Ni J., Dixit V. M. RIP2 is a novel NF-{kappa}B-activating and cell death-inducing kinase. J. Biol. Chem., 273: 16968-16975, 1998.[Abstract/Free Full Text]
  25. Bertin J., Nir W-J., Fischer C. M., Tayber O. V., Errada P. R., Grant J. R., Keilty J. J., Gosselin M. L., Robison K. E., Wong G. H. W., Glucksmann M. A., DiStefano P. S. Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-{kappa}B. J. Biol. Chem., 274: 12955-12958, 1999.[Abstract/Free Full Text]
  26. Inohara N., Koseki T., del Peso L., Hu Y., Yee C., Chen S., Carrio R., Merino J., Liu D., Ni J., Nunez G. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-{kappa}B. J. Biol. Chem., 274: 14560-14567, 1999.[Abstract/Free Full Text]
  27. Koseki T., Inohara N., Chen S., Carrio R., Merino J., Hottiger M. O., Nabel G. J., Nunez G. CIPER, a novel NF-{kappa}B-activating protein containing a caspase recruitment domain with homology to herpesvirus-2 protein E10. J. Biol. Chem., 274: 9955-9961, 1999.[Abstract/Free Full Text]
  28. Srinivasula S. M., Ahmad M., Lin J-h., Poyet J-L., Fernandes-Alnemri T., Tsichlis P. N., Alnemri E. S. CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-{kappa}B activation and apoptosis. J. Biol. Chem., 274: 17946-17954, 1999.[Abstract/Free Full Text]
  29. Baichwal V. R., Baeuerle P. A. Apoptosis. activate NF-{kappa}B or die?. Curr. Biol., 7: R94-R96, 1997.[Medline]
  30. Wang C-Y., Mayo M. W., Korneluk R. G., Goeddel D. V., Baldwin A. S., Jr. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science (Washington DC), 281: 1680-1683, 1998.[Abstract/Free Full Text]
  31. Pan H., Yin C., Van Dyke T. Apoptosis and cancer mechanisms. Cancer Surv., 29: 305-327, 1997.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
E. de Alba
Structure and Interdomain Dynamics of Apoptosis-associated Speck-like Protein Containing a CARD (ASC)
J. Biol. Chem., November 20, 2009; 284(47): 32932 - 32941.
[Abstract] [Full Text] [PDF]

Home page
 Anticancer ResHome page
E. GORDIAN, K. RAMACHANDRAN, and R. SINGAL
Methylation Mediated Silencing of TMS1 in Breast Cancer and its Potential Contribution to Docetaxel Cytotoxicity
Anticancer Res, August 1, 2009; 29(8): 3207 - 3210.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J. Parsons, P. Patel, D. J. Brat, L. Colbert, and P. M. Vertino
Silencing of TMS1/ASC Promotes Resistance to Anoikis in Breast Epithelial Cells
Cancer Res., March 1, 2009; 69(5): 1706 - 1711.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. T.-H. Huang, D. J. Taxman, E. A. Holley-Guthrie, C. B. Moore, S. B. Willingham, V. Madden, R. K. Parsons, G. L. Featherstone, R. R. Arnold, B. P. O'Connor, et al.
Critical Role of Apoptotic Speck Protein Containing a Caspase Recruitment Domain (ASC) and NLRP3 in Causing Necrosis and ASC Speck Formation Induced by Porphyromonas gingivalis in Human Cells
J. Immunol., February 15, 2009; 182(4): 2395 - 2404.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
P. Kapoor-Vazirani, J. D. Kagey, D. R. Powell, and P. M. Vertino
Role of hMOF-Dependent Histone H4 Lysine 16 Acetylation in the Maintenance of TMS1/ASC Gene Activity
Cancer Res., August 15, 2008; 68(16): 6810 - 6821.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
N. Yajima, M. Takahashi, H. Morimoto, Y. Shiba, Y. Takahashi, J. Masumoto, H. Ise, J. Sagara, J. Nakayama, S. Taniguchi, et al.
Critical Role of Bone Marrow Apoptosis-Associated Speck-Like Protein, an Inflammasome Adaptor Molecule, in Neointimal Formation After Vascular Injury in Mice
Circulation, June 17, 2008; 117(24): 3079 - 3087.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. O. Machida, M. V. Brock, C. M. Hooker, J. Nakayama, A. Ishida, J. Amano, M. A. Picchi, S. A. Belinsky, J. G. Herman, S. Taniguchi, et al.
Hypermethylation of ASC/TMS1 Is a Sputum Marker for Late-Stage Lung Cancer.
Cancer Res., June 15, 2006; 66(12): 6210 - 6218.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Sarkar, M. Duncan, J. Hart, E. Hertlein, D. C. Guttridge, and M. D. Wewers
ASC Directs NF-{kappa}B Activation by Regulating Receptor Interacting Protein-2 (RIP2) Caspase-1 Interactions.
J. Immunol., April 15, 2006; 176(8): 4979 - 4986.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. J. Kaiser and M. K. Offermann
Apoptosis Induced by the Toll-Like Receptor Adaptor TRIF Is Dependent on Its Receptor Interacting Protein Homotypic Interaction Motif
J. Immunol., April 15, 2005; 174(8): 4942 - 4952.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
M. Yamamoto, K. Yaginuma, H. Tsutsui, J. Sagara, X. Guan, E. Seki, K. Yasuda, M. Yamamoto, S. Akira, K. Nakanishi, et al.
ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules
Genes Cells, November 1, 2004; 9(11): 1055 - 1067.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
C. Stehlik and J. C. Reed
The PYRIN Connection: Novel Players in Innate Immunity and Inflammation
J. Exp. Med., September 7, 2004; 200(5): 551 - 558.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Kubo, J. Yamamoto, Y. Shikauchi, Y. Niwa, K. Matsubara, and H. Yoshikawa
Apoptotic Speck Protein-Like, a Highly Homologous Protein to Apoptotic Speck Protein in the Pyrin Domain, Is Silenced by DNA Methylation and Induces Apoptosis in Human Hepatocellular Carcinoma
Cancer Res., August 1, 2004; 64(15): 5172 - 5177.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
K. Terasawa, S. Sagae, M. Toyota, K. Tsukada, K. Ogi, A. Satoh, H. Mita, K. Imai, T. Tokino, and R. Kudo
Epigenetic Inactivation of TMS1/ASC in Ovarian Cancer
Clin. Cancer Res., March 15, 2004; 10(6): 2000 - 2006.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Garcia-Domingo, D. Ramirez, G. Gonzalez de Buitrago, and C. Martinez-A
Death Inducer-Obliterator 1 Triggers Apoptosis after Nuclear Translocation and Caspase Upregulation
Mol. Cell. Biol., May 1, 2003; 23(9): 3216 - 3225.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
C. Stehlik, L. Fiorentino, A. Dorfleutner, J.-M. Bruey, E. M. Ariza, J. Sagara, and J. C. Reed
The PAAD/PYRIN-Family Protein ASC Is a Dual Regulator of a Conserved Step in Nuclear Factor {kappa}B Activation Pathways
J. Exp. Med., December 16, 2002; 196(12): 1605 - 1615.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. M. Stimson and P. M. Vertino
Methylation-mediated Silencing of TMS1/ASC Is Accompanied by Histone Hypoacetylation and CpG Island-localized Changes in Chromatin Architecture
J. Biol. Chem., February 8, 2002; 277(7): 4951 - 4958.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Richards, P. Schaner, A. Diaz, J. Stuckey, E. Shelden, A. Wadhwa, and D. L. Gumucio
Interaction between Pyrin and the Apoptotic Speck Protein (ASC) Modulates ASC-induced Apoptosis
J. Biol. Chem., October 12, 2001; 276(42): 39320 - 39329.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
J. Masumoto, S.'i. Taniguchi, J. Nakayama, M. Shiohara, E. Hidaka, T. Katsuyama, S. Murase, and J. Sagara
Expression of Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain, a Pyrin N-terminal Homology Domain-containing Protein, in Normal Human Tissues
J. Histochem. Cytochem., October 1, 2001; 49(10): 1269 - 1276.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
J. F Costello and C. Plass
Methylation matters
J. Med. Genet., May 1, 2001; 38(5): 285 - 303.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McConnell, B. B.
Right arrow Articles by Vertino, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McConnell, B. B.
Right arrow Articles by Vertino, P. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online