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

Qin Chen, Victoria C. Tu, Juping Liu

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 β-galactosidase (SA β-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 β-gal until 4 days after the treatment with 75 μM 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 β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 μM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

Original languageEnglish (US)
Pages (from-to)335-339
Number of pages5
JournalBiogerontology
Volume1
Issue number4
StatePublished - 2000

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Galactosidases
Diploidy
Hydrogen Peroxide
Fibroblasts
DNA
Serial Passage
Retinoblastoma Protein
Cell Aging
Oxidants

Keywords

  • Dose response
  • Growth arrest
  • Oxidants
  • Senescent biomarkers

ASJC Scopus subject areas

  • Geriatrics and Gerontology
  • Gerontology
  • Aging

Cite this

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title = "Measurements of hydrogen peroxide induced premature senescence: Senescence-associated β-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 β-galactosidase (SA β-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 β-gal until 4 days after the treatment with 75 μM 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 β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 μM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.",
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AU - Tu, Victoria C.

AU - Liu, Juping

PY - 2000

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N2 - 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 β-galactosidase (SA β-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 β-gal until 4 days after the treatment with 75 μM 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 β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 μM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

AB - 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 β-galactosidase (SA β-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 β-gal until 4 days after the treatment with 75 μM 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 β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 μM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

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