Promoter hypermethylation of the ADAM23 gene in colorectal cancer cell lines and cancer tissues

Abstract

Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell-to-cell and cell-to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation-specific PCR (MSP) and bisulfate-modified DNA sequencing analysis. Methylated cells were treated with 5-aza-2 0 -deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70-90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5-aza-2 0 -deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down-regulated by aberrant promoter hypermethylation during the progression of colorectal cancer. ' 2008 Wiley-Liss, Inc.Key words: ADAM23 gene; methylation; methylation-specific PCR (MSP); colorectal cancer; cancer cell lines Colorectal cancer is the third most common cancer in both males and females in the United States, although incidence rates have decreased from 1998 through 2004. 1 In Korea, colorectal cancer is the fourth common cancer in both sexes. 2 Moreover, evidence indicates that most cancers have a genetic component. 3 However, it is also believed that epigenetic mechanisms may also have a significant role in colorectal cancer development, 4 and methylation of the C 5 position of cytosine residues in DNA is one of the most fundamental epigenetic characteristics. This methylation is performed by DNA methyltransferases (DNMTs), which have been implicated in many processes including transcriptional regulation, genomic stability, X-chromosome inactivation and in the silencing of parasitic DNA transposable elements. 5 In a recent study, it was estimated that up to 80% of all CpG dinucleotides in the genome are methylated. 6 Furthermore, unmethylated CpG residues are known to be predominantly located in the promoter regions of active and inducible genes. They are referred to as CpG islands, 7 which consist of regions of more than 500 base pairs with the GC content exceeding 55%. 6 The importance of DNA methylation was highlighted by the finding that many human disease result from abnormal control. 8 Moreover, the aberrant methylation of CpG islands is a characteristic of many human cancers and may be found during early carcinogenesis. 9 This methylation may silence many genes, esp...

Matrix metalloproteinase 2 promotes cell growth and invasion in colorectal cancer

“…Genetic alterations, together with epigenetic components and dietary factors, play a fundamental role in the initiation and progression of CRC by causing a number of deregulations, such as the activation of oncogenes and inactivation of tumor suppressors [22][23][24][25][26][27]. However, the molecular mechanism of CRC pathogenesis remains incompletely understood.…”

CHFR Promoter Methylation Indicates Poor Prognosis in Stage II Microsatellite Stable Colorectal Cancer

“…Promoter CpG island methylation of genes reported to be methylated in CRC (2,5,22,23): mutL homolog1, colon cancer, nonpolyposis type 2 (Escherichia coli; MLH1), cyclin-dependent kinase inhibitor 2A (CDKN2A; p16INK4 and p14ARF), O-6-methylguanine-DNA methyltransferase (MGMT), Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A), adenomatous polyposis coli (APC), helicase-like transcription factor (HLTF), GATA-binding protein 4 (GATA4), GATA-binding protein 5 (GATA5), checkpoint with forkhead and ring finger domains (CHFR), ADAM metallopeptidase domain 23 (ADAM23), Rab32, member RAS oncogene family (RAB32), junctophilin (JPH3), forkhead box L2 (FOXL2), BCL2-adenovirus E1B 19kDA interacting protein 3 (BNIP3), neutralized homolog (Drosophila; NEURL), calcium channel, voltage dependent, a2-delta subunit 1 (CACNA2), thrombospondin 1 (THBS1), tissue factor pathway inhibitor 2 (TFPI2), and the CIMP genes calcium channel, voltage-dependent, T type, a-1G subunit (CACNA1G), insulin-like growth factor-II (somatomedin A, IGF-II), neurogenin 1 (NEUROG1), runt-related transcription factor 3 (RUNX3), and suppressor of cytokine signaling 1 (SOCS1) were determined using sodium bisulfite modification of genomic DNA (EZ DNA Methylation Kit, ZYMO research Co.). To facilitate methylation-specific PCR (MSP) analysis on DNA retrieved from formalinfixed, paraffin-embedded tissue, nested MSP was performed as described elsewhere (24,25).…”

TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas

“…For bisulfite treatment, 1 g genomic DNA was used, digested with BamHI as previously described. 16 The primer sequences for methylationspecific PCR (MSP) are described in supplemental Table 3. The PCR conditions consisted of 10 minutes at 95°C for initial denaturation, followed by 35 cycles at 95°C (30 seconds), 54°C (30 seconds), and 72°C (30 seconds) and a final extension of 10 minutes at 72°C using AmpliTaq Gold DNA polymerase (Applied Biosystems).…”