Measurements of hydrogen peroxide induced premature senescence: senescence-associated beta-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb.

Abstract

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated beta-galactosidase (SA beta-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA beta-gal until 4 days after the treatment with 75 microM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA beta-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA beta-gal one week following the treatment with H2O2 at doses lower than 150 microM or 1.5 pmol/cell. The dose response curve of SA beta-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA beta-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.