Chromium (VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest, DNA-DNA cross-links and S-phase blockade of cell cycle

Jian Xu, Glenn J. Bubley, Barbara Detrick, Lori J. Blankenship, Steven R. Patierno

Research output: Contribution to journalArticle

Abstract

Previous studies have shown that in vitro treatment of a synthetic double-stranded DNA template with chromium (III), or chromium (VI) in the presence of ascorbate, resulted in guanine-specific DNA polymerase arrests that correlated strongly with DNA-DNA cross-linking. In vivo chromium (VI) undergoes a more complicated intracellular cascade of reductive metabolism than is achievable in an in vitro model. Moreover, in living cells, DNA is highly packaged in the form of chromatin which may alter the accessibility of DNA to chromium. A repetitive primer-extension assay was employed to determine whether chromium forms polymerase-arresting lesions in vivo. Normal human lung fibroblasts treated with chromium.(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucleotides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA interstrand cross-links. Genomic DNA was isolated and alphoid sequences (1-5% of the genome) were used as a substrate for repetitive primer extension using Tag polymerase. The results showed a dose-dependent, guanine-specific, replication termination, even at low doses resulting in greater than 90% survival. The same treatment resulted in a dose-dependent suppression of thymidine incorporation into DNA immediately after treatment. Thymidine incorporation increased during the first 6 h after the 2-h exposure, probably related to the repair of single strand breaks, but then returned to high suppression levels at 24 h. The chromate treatments inhibited cell growth by specific blocking of the progression of cells through S-phase of the cell cycle. The results confirmed our studies in cell-free systems and taken together they strongly indicate that guanine-guanine DNA interstrand cross-links induced by chromate in living cells is the lesion responsible for blocking DNA replication processivity.

Original languageEnglish (US)
Pages (from-to)1511-1517
Number of pages7
JournalCarcinogenesis
Volume17
Issue number7
DOIs
StatePublished - Jul 1996
Externally publishedYes

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Guanine
DNA-Directed DNA Polymerase
S Phase
Cell Cycle
Lung
DNA
Chromium
Chromates
Thymidine
chromium hexavalent ion
Genome
Cell-Free System
DNA Adducts
DNA Replication
Chromatin
Nucleotides
Fibroblasts
Survival

ASJC Scopus subject areas

  • Cancer Research

Cite this

Chromium (VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest, DNA-DNA cross-links and S-phase blockade of cell cycle. / Xu, Jian; Bubley, Glenn J.; Detrick, Barbara; Blankenship, Lori J.; Patierno, Steven R.

In: Carcinogenesis, Vol. 17, No. 7, 07.1996, p. 1511-1517.

Research output: Contribution to journalArticle

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abstract = "Previous studies have shown that in vitro treatment of a synthetic double-stranded DNA template with chromium (III), or chromium (VI) in the presence of ascorbate, resulted in guanine-specific DNA polymerase arrests that correlated strongly with DNA-DNA cross-linking. In vivo chromium (VI) undergoes a more complicated intracellular cascade of reductive metabolism than is achievable in an in vitro model. Moreover, in living cells, DNA is highly packaged in the form of chromatin which may alter the accessibility of DNA to chromium. A repetitive primer-extension assay was employed to determine whether chromium forms polymerase-arresting lesions in vivo. Normal human lung fibroblasts treated with chromium.(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucleotides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA interstrand cross-links. Genomic DNA was isolated and alphoid sequences (1-5{\%} of the genome) were used as a substrate for repetitive primer extension using Tag polymerase. The results showed a dose-dependent, guanine-specific, replication termination, even at low doses resulting in greater than 90{\%} survival. The same treatment resulted in a dose-dependent suppression of thymidine incorporation into DNA immediately after treatment. Thymidine incorporation increased during the first 6 h after the 2-h exposure, probably related to the repair of single strand breaks, but then returned to high suppression levels at 24 h. The chromate treatments inhibited cell growth by specific blocking of the progression of cells through S-phase of the cell cycle. The results confirmed our studies in cell-free systems and taken together they strongly indicate that guanine-guanine DNA interstrand cross-links induced by chromate in living cells is the lesion responsible for blocking DNA replication processivity.",
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