Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines.
Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.
Honoki
Kanya
K
Department of Orthopedic Surgery, Nara Medical University, Kashihara, Nara, Japan.
Tsujiuchi
Toshifumi
T
Mori
Toshio
T
Yoshitani
Kazuhiro
K
Tsutsumi
Masahiro
M
Takakura
Yoshinori
Y
Mii
Yoshio
Y
D000230:Adenocarcinoma / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018899:CpG Islands / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D000077209:Decitabine; D005347:Fibroblasts / Q000473:pathology; D020868:Gene Silencing; D018219:Histiocytoma, Benign Fibrous / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics; D009369:Neoplasms / Q000235:genetics*; D012516:Osteosarcoma / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013447:Sulfites; D016385:TATA Box; D014407:Tumor Cells, Cultured
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger; D013447:Sulfites; D000077209:Decitabine; D001374:Azacitidine; C009279:sodium bisulfite