Cancer Letters

Cancer Letters

Volume 202, Issue 1, 8 December 2003, Pages 101-108
Cancer Letters

Methylation of ASC/TMS1, a proapoptotic gene responsible for activating procaspase-1, in human colorectal cancer

https://doi.org/10.1016/j.canlet.2003.08.027Get rights and content

Abstract

ASC/TMS1, a proapoptotic activator of procaspase-1, was reported to be aberrantly methylated in human breast cancer. We found that ASC was methylated in three of five human colon cancer cell lines lacking ASC protein expression. Demethylation treatment of these cell lines lacking ASC with 5-aza-2′-deoxycytidine partially restored ASC expression. Methylated ASC was also detected in six of ten colorectal cancer tissues. Although clear down-regulation of ASC in the whole region of a tumor tissue was hardly observed by immunostaining with anti-ASC mAb, complete suppression of ASC was identified in a minor population of the colorectal tumor cells. The biological significance of ASC methylation inducible ASC suppression in colorectal cancer will be discussed.

Introduction

While genetic approaches to understanding cancer have made great progress, directed attention to epigenetic events has been increasing [1]. Aberrant methylation of CpG islands, as one epigenetic event, is detected relatively frequently in transformed cells, in particular in association with the silencing of certain human suppressor genes [1], [2], [3].

ASC, a 22-kDa protein, is a member of the CED4/Apaf1 family cloned by our group in 1999 [4], [5]. This protein was found forming speck-like aggregations during the process of retinoic acid-induced neutrophilic differentiation and apoptosis of the promyeloleukemic cell line, HL-60 [5]. Its carboxyl terminal has a caspase recruitment domain (CARD) motif [6], characteristic of numerous proteins involved in apoptotic signaling, while its amino terminal has a domain structure homologous to that of the Pyrin (PYD) [7], [8], [9], a protein involved in the inflammatory disorder known as familial Mediterranean fever [10]. In addition to the pro-apoptotic function, ASC has been proved to be an adaptor protein-activating procaspase-1 that is necessary in generating IL-1β [11] and IL-18 (IFN-γ-inducing factor) [12], which are known as pro-inflammatory cytokines via cleavage of their pro-forms. IL-18 has also been reported to exhibit in vivo immunologically mediated anti-tumor effects in mice [13], [14].

Recently, Conway et al. reported inhibition of ASC/TMS1 gene expression by methylation in human breast cancer, suggesting a direct association between methylation and carcinogenesis [15]. They suggested that ASC/TMS1 is anti-oncogenic through its pro-apoptotic function. The methylation of CpG islands in ASC/TMS1 proved to result in hypoacetylation of histones H3 and H4, inhibiting transcription [16].

In colorectal and other tumors, methylation in the promoter region of the APC gene constitutes an alternative mechanism for gene inactivation [17]. The hypermethylation of CACNA1G, T-type calcium channel gene that is involved in cell proliferation and cell death, was observed and CACNA1G expression was absent in human colorectal tumors [18]. In this study, we investigated the relationship between ASC expression and methylation in colorectal cancers.

Section snippets

Cell lines

Experiments were conducted with five human colorectal cancer cell lines, COLO201, COLO320DM, DLD-1, LoVo and WiDr cells. These were obtained from the Health Science Research Resources Bank (Osaka, Japan). Stock cultures were maintained in the following media: RPMI1640 (Nihon seiyaku, Tokyo, Japan: COLO201 and DLD-1); Dulbecco's modified Eagle's medium (Nihon seiyaku: COLO320DM and WiDr); Ham's F-12 (Nihon seiyaku: LoVo). These media were supplemented with 10% (COLO201, COLO320DM, DLD-1 and

Expression of ASC in human colorectal cancer cell lines

Expression of ASC protein in five colorectal cancer cell lines was examined by Western blot analysis. ASC expression was detected in COLO201 and WiDr, but not in COLO320DM, DLD-1 or LoVo cells (Fig. 1).

DNA methylation status of the ASC gene in human colorectal cancer cell lines

The DNA methylation status of the ASC gene in human colorectal cancer cell lines was assessed by the MSP technique in which methylated and unmethylated alleles can be specifically amplified after chemical modification of the DNA with sodium bisulfite. As shown in Fig. 2, ASC was completely

Discussion

ASC protein was detected in COLO201 and WiDr, but not in completely or partially methylated COLO320DM, DLD-1 and LoVo, indicating that presence or absence of ASC protein suppression was well correlated with unmethylation or methylation of a CpG island in the ASC gene, while, the treatment of DNA methyltransferase inhibitor resulted in slight but significant restoration of ASC protein expression. In the case of LoVo, the cell line has unmethylated ASC, so that the down-regulation of ASC protein

Acknowledgements

We thank Mr Satoshi Nakano (Jisen-kai Aizawa hospital) for his technical assistance.

References (30)

  • Z.L. Chu et al.

    A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis

    J. Biol. Chem.

    (2001)
  • B.J. Geddes et al.

    Human CARD12 is a novel CED4/Apaf-1 family member that induces apoptosis

    Biochem. Biophys. Res. Commun.

    (2001)
  • J.L. Poyet et al.

    Identification of Ipaf, a human caspase-1-activating protein related to Apaf-1

    J. Biol. Chem.

    (2001)
  • S.M. Srinivasula et al.

    The PYRIN-CARD protein ASC is an activating adaptor for caspase-1

    J. Biol. Chem.

    (2002)
  • M. Toyota et al.

    CpG island methylator phenotype in colorectal cancer

    Proc. Natl Acad. Sci. USA

    (1999)
  • Cited by (54)

    • The NLRP1 Inflammasome Pathway Is Silenced in Cutaneous Squamous Cell Carcinoma

      2019, Journal of Investigative Dermatology
      Citation Excerpt :

      Reports about the role of inflammasomes in cancer are controversial (Karki et al., 2017; Kolb et al., 2014). As mentioned before, the expression of the inflammasome protein ASC is silenced in different cancer types, including dedifferentiated SCCs, melanoma, and others (Meier et al., 2016; Wu et al., 2016; Yokoyama et al., 2003). The expression levels of inflammasome proteins are down-regulated in skin carcinoma-derived SCC cell lines in vitro (Figure 1).

    • Transcriptional regulation of inflammasome-associated pattern recognition receptors, and the relevance to disease pathogenesis

      2017, Molecular Immunology
      Citation Excerpt :

      While the above studies identify genetic alterations to inflammasome-related gene promoters which influence their transcriptional activity in cancer, it is also noteworthy that epigenetic mechanisms involving DNA methylation can influence the gene expression of inflammasome-associated components in cancer. This is best evidenced for ASC, which is transcriptionally suppressed by hypermethylation of a CpG island within its promoter region in numerous cancers, including renal, colorectal and ovarian (Liu et al., 2015a,b; Terasawa et al., 2004; Yokoyama et al., 2003). Conversely, in acute lymphoblastic leukemia, resistance to glucocorticoid treatment has been associated with hypomethylation of the NLRP3 (and CASP1) gene promoters, resulting in elevated expression of these genes, as well as inflammasome activity (Paugh et al., 2015).

    • Cancer Pathogenesis and DNA Sensing

      2013, Biological DNA Sensor: The Impact of Nucleic Acids on Diseases and Vaccinology
    View all citing articles on Scopus
    View full text