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CpG methylation of the PAI-1 gene 5'-flanking region is inversely correlated with PAI-1 mRNA levels in human cell lines.

The physiological and pathophysiological functions of PAI-1 are related to its expression by specific cell types in normal and diseased tissues. We analysed the contribution of DNA methylation to the variation in PAI-1 mRNA levels in five cell lines. We found varying frequencies of methylation of 25 CpGs in the -805/+152 region of the PAI-1 gene in Bowes, MCF-7 and U937 cells, while little or no methylation was detected in Hep2 and HT- 1080 cells. The methylation frequency was inversely correlated with PAI-1 mRNA level within its 20-fold range in Bowes, MCF-7, U937,and Hep2 cells, while the lack of methylation in both Hep2 and HT-1080 cells suggested another mechanism behind the 150-fold higher level in HT- 1080 cells than in Hep2 cells. However, all cell lines exhibited a high frequency of methylation of 10 CpGs in a CpG island at about--1800. Treatment with 5-aza-2'-deoxycytidine led up to circa a 40-fold increase in the PAI-1 mRNA level and a strong decrease in the frequency of methylation in the -805/+152 region in Bowes, MCF-7 and U937. The histone deacetylase inhibitor trichostatin A induced a several fold increase of the PAI-I mRNA level in cells with a high methylation frequency of the -805/+152 region. As compared with matched normal tissue, three samples of oral squamous cell carcinomas displayed decreased frequencies of methylation of the PAI-1 5' flanking region and increased levels of PAI-1 mRNA. These results for the first time implicate DNA methylation and histone acetylation in regulation of the PAI-1 gene, and indicate that without proper CpG islands in 5'-flanking region,trancription may be regulated by methylation of less dense CpGs in the 5'-flanking region rather than methylation of upstream CpG island.