Naslov
IGF2 promoter usage and IGF2AS expression in tumor cell lines Cal27 and HT-29

Autori
Grbeša, Ivana ; Kolundžija, Sandra ; Novak Kujundžić, Renata ; Gall-Trošelj, Koraljka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, neobjavljeni rad, znanstveni

Skup
2nd Annual Meeting on Cancer and Control of Genomic Integrity 2009

Mjesto i datum
Stockholm, Švedska, 21.08.2009. - 23.08.2009

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
IGF2 ; IGF2AS

Sažetak
The transcription of imprinted IGF2 is regulated by five promoters in a tissue-and age-specific manner. Its antisense transcript originates from the promoter located within a CpG island near IGF2 promoters P2-P4. The IGF-Bi fragment in this region has insulator properties and contains methylation-sensitive CTCF-binding sites. Our aim was to determine the influence of DNA methylation and ADP-ribosylation on the regulation of IGF2 promoter usage and IGF2AS transcription in two human tumor cell lines. They were chosen based on their different origin, Cal27 (tongue– mesodermal) and HT-29 (colon-endodermal) considering the activation of IGF2 by mesodermal or endodermal enhancers. Cells were treated with PARP-1 inhibitor, 3-aminobenzamide (3-AB), to inhibit poly(ADP-ribosyl)ation and promote DNA methylation, or 5-azacytidine (5-aza) to induce DNA demethylation and inhibit poly(ADP-ribosyl)ation. DNA methylation of IGF2AS promoter was analyzed by COBRA. Expression of IGF2AS was analyzed by real-time PCR. The IGF2 promoter usage was analyzed by RT-PCR. Both untreated cell lines had hypomethylated IGF2AS promoter to which CTCF could bind, but IGF2AS was expressed only in HT-29 cells. The IGF2 promoter usage differed: in HT-29 cells all five IGF2 promoters were active, while in Cal27 cells IGF2 originated only from promoters P3 and P4. Treatment with 5-aza downregulated IGF2AS expression in HT-29 cells by 60% and silenced IGF2 transcription from P2. Treatment with 3-AB downregulated IGF2AS expression in HT-29 cells by 30% concomitant with downregulation of IGF2 P2 transcription to undetectable level. Methylation status of IGF2AS promoter and transcription of IGF2 from other promoters was unchanged. Both treatments had no effect on IGF2AS promoter methylation, its expression or IGF2 promoter usage. Our results suggest that: 1) IGF2AS expression coincides with IGF2 expression from P1 and P0 ; 2) binding of CTCF to hypomethylated IGF2-Bi element prevents transcription from P1, P0 and P2 only in mesoderm-derived Cal27, but not endoderm-derived HT29 cells.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti

POVEZANOST RADA