DNA-binding proteins in young and senescent normal human fibroblasts

Abstract

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either 3H- or 14C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.