Repression by hnRNP-K of the CD43 promoter present within chromosomal DNA.

K562 cells were transfected with the linearized plasmid p43Wt/Zeo, which was derived from p43Wt by insertion of thezeocin-resistance gene. K562 cells in which the p43Wt/Zeo plasmid was stably integrated within the genome were selected by treatment with zeocin. The mixed pool of zeocin-resistant cells was then transfected with 2 μg of pRSV-β mixed with either 23 μg of full-length hnRNP-K or 23 μg of its parent vector empty ofhnRNP-K–coding sequences. Transfections were also performed using 9 μg of pRSV-β mixed with either 16 μg of full-length hnRNP-K or 16 μg of its parent. In those experiments in which the role of DNA methylation was assessed, cells were either untreated or pretreated for 48 hours with 5-azacytidine prior to transfection with 2 μg of pRSV-β mixed with either 23 μg of full-length hnRNP-K or 23 μg of its parent. Following all transfections, cells were treated with PMA for 12 hours prior to harvesting and assay of luciferase and β-galactosidase activities. In experiments assessing the influence of DNA methylation, transfected cells, untreated or pretreated with 5-azacytidine, were treated simultaneously with PMA and 5-azacytidine for 12 hours prior to harvesting. The levels of β-galactosidase activity were taken as reflective of transfection efficiency and used to correct the luciferase assay results. The level of luciferase activity directed by the CD43 promoter in the presence of the vector empty of hnRNP-K coding sequences (−hnRNP-K) was assigned a value of 100%. The luciferase levels directed by theCD43 promoter in matched, parallel transfections using full-length hnRNP-K (+hnRNP-K) were calculated as a proportion of the 100% value. The means of these proportional values ± SD resulting from 3 independent experiments are displayed.