The methylation specific PCR in conjunction of sequencing verification was used to establish the methylation-profile of the promoter CpG islands of 31 genes in
colorectal cancer (n = 65), the neighboring non-cancerous tissues (n = 5), colorectal
adenoma (n = 8), and normal mucosa (n = 1). Immunohistochemically, expression of 10 genes was assessed on the home-made tissue microarrays of tissues from 58 patients. The correlation of
tumor specific changes with each of clinical-pathologic features was scrutinized with relevant statistic tools.
RESULTS: In comparison with the normal mucosa of the non-
cancer patients, the following 14 genes displayed no
tumor associated changes:
breast cancer 1, early onset (BRCA1),
cadherin 1, type 1,
E-cadherin (epithelial) (CDH1),
death-associated protein kinase 1 (DAPK1),
DNA (cytosine-5-)-methyltransferase 1 (DNMT1),
melanoma antigen, family A, 1 (directs expression of
antigen MZ2-E) (MAGEA1),
tumor suppressor candidate 3 (N33),
cyclin-dependent kinase inhibitor 1A (p21, Cip1) (p21(WAF1)),
cyclin-dependent kinase inhibitor 1B (p27, Kip1) (p27(KIP1)),
phosphatase and
tensin homolog (mutated in multiple advanced
cancers 1) (PTEN),
retinoic acid receptor, beta (RAR- , Ras association (
RalGDS/AF-6) domain family 1 C (RASSF1C),
secreted frizzled-related protein 1 (SFRP1),
tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) (TIMP3), and
von Hippel-Lindau syndrome (VHL). The rest 17 targets exhibited to various extents the
tumor associated changes. As changes in methylation of the following genes occurred marginally, their impact on the formation of
colorectal cancer were trivial:
adenomatous polyposis coli (APC) (8%, 5/65), Ras association (
RalGDS/AF-6) domain family 1A (RASSF1A) (3%, 2/65) and
cyclin-dependent kinase inhibitor 2A, alternated reading frame (
p14(ARF)) (6%, 4/65). The following genes exhibited moderate changes in methylation: O-6-methylguanine-
DNA methyltransferase (MGMT) (20%, 13/65),
mutL homolog 1,
colon cancer, nonpolyposis type 2 (E. coli) (hMLH1) (18%, 12/65),
cyclin-dependent kinase inhibitor 2A (
melanoma, p16, inhibits CDK4) (
p16(INK4a)) (10%, 10/65), methylated in
tumor 1 (MINT1) (15%, 10/65), methylated in
tumor 31 (MINT31) (11%, 7/65). The rest changed greatly in the methylation pattern in
colorectal cancer (CRC):
cyclin A1 (
cyclin a1) (100%, 65/65), caudal type homeobox
transcription factor 1 (CDX1) (100%, 65/65), RAR- (85%, 55/65), myogenic factor 3 (MYOD1) (69%, 45/65),
cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (p15(INK4b)) (68%, 44/65),
prostaglandin-endoperoxide synthase 2 (
prostaglandin G/H synthase and
cyclooxygenase) (COX2) (72%, 47/65),
cadherin 13,
H-cadherin (heart) (CDH13) (65%, 42/65), CAAX box 1 (CXX1) (58%, 38/65),
tumor protein p73 (p73) (63%, 41/65) and
Wilms tumor 1 (WT1) (58%, 38/65). However, no significant correlation of changes in methylation with any given clinical-pathological features was detected. Furthermore, the frequent changes in methylation appeared to be an early phase event of colon
carcinogenesis. The in situ expression of 10 genes was assessed by the immunohistochemical approach at the
protein level: CDH1, CDH13, COX2,
cyclin A1, hMLH1, MGMT,
p14(ARF), p73, RAR- , and TIMP3 genes in the context of the methylation status in
colorectal cancer. No clear correlation between the hypermethylation of the promoter CpG islands and the negative expression of the genes was established.
CONCLUSION: