Changes in methylation pattern of albumin and α-fetoprotein genes in developing rat liver and neoplasia

Abstract

To determine whether methylation changes in specific DNA sequences of the albumin and AFP genes are implicated in the modulation of transcriptional activity during rat liver development and neoplasia we have analysed the methylation pattern of C-C-G-G sequences within these genes in DNA isolated from fetal and adult hepatocytes, from adult kidney and from a clonal hepatoma cell line which produces AFP but no albumin. We have assayed for methylation of the internal cytosine of this sequence by using the restriction enzyme isoschizomers HpaII and MspI. 32P-labelled cloned cDNA probes were used to reveal the albumin and AFP gene containing fragments. Genomic subclones of the albumin gene were also utilized as molecular probes to measure quantitatively the level of methylation of 6 specific sites within the albumin gene in the different DNA samples. The results indicate that methylation changes at the sites analysed are not responsible for the changes in gene activity during rat liver development. Further they demonstrate that: 1) extensively methylated genes can be actively transcribed; 2) prominent changes in methylation of specific genes during normal development are not necessarily related to alterations in gene activity.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

“…SC). The AlbIV and AlbV sites are found about 3.5 and 5 kb, (19,33,47). Comparison of an AFP-expressing hepatoma cell line and a nonexpressing one led to the conclusion that some specific intragenic sites might be undermethylated when the gene is expressed (20,47).…”

“…The AlbIV and AlbV sites are found about 3.5 and 5 kb, (19,33,47). Comparison of an AFP-expressing hepatoma cell line and a nonexpressing one led to the conclusion that some specific intragenic sites might be undermethylated when the gene is expressed (20,47). Concerning the albumin gene, parallel analysis of albuminexpressing and nonexpressing hepatoma cell lines showed that the demethylation of a cytosine located in exon 1 of the albumin gene is well correlated with gene expression (29,30).…”

“…We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaH Many studies with different experimental systems have been carried out to elucidate the role that methylation of cytosine bases and change in chromatin structure might play in the regulation of gene expression (for reviews, see references 34 and 35).Controversial results concerning the relationship between the DNA methylation pattern and gene activity have been reported showing either a good correlation (23,47) or no correlation at all (13,46,57) between undermethylation and expression of a specific gene. Various studies involving in vitro methylation of a specific regulatory region of a gene followed by its introduction into eucaryotic cells revealed that DNA methylation does affect transcription (6,17,41,42).…”

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes

“…Heypatocytes were isolated from Wistar rats by collagenase perfusion technique (Seglen,3 979). The isolation of a Morris hepatoma 7777 cell line has been described by Vedel et al (1983). All cells were maintained at 37' C and 5% COz.…”

Spontaneous and 5-azacytidine-induced reexpression of ornithine carbamoyl transferase in hepatoma cells.

“…On the other hand, modifications (increase or decrease) in the general level of DNA methylation (19,27) or in the methylation of specific genes (9,25,26,40,42) of tumor cells have been reported. Changes in DNA methylation have been proposed to play an important role in the abnormally regulated expression of tumor cell genes (8,14).…”