Cytotoxicity and DNA fragmentation associated with sequential gemcitabine and 5-fluoro-2'-deoxyuridine in HT-29 colon cancer cells

Qianfang Ren, Vivian Kao, Jean L. Grem

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

The combined cytotoxic effects of the antimetabolites gemcitabine (dFdCyd) and 5-fluoro-2'-deoxyuridine (FdUrd) were studied. Cytotoxicity, biochemical perturbations, and DNA damage seen with dFdCyd and FdUrd alone and in combination were evaluated in HT-29 human colon cancer cells. A 4-h exposure to dFdCyd followed by FdUrd for 24 h produced more than additive cytotoxicity and marked S-phase accumulation. Cells progressed through the cell cycle, however, after a 22-h drug-free interval. [3H]dFdCyd was rapidly metabolized to the 5'-triphosphate and incorporated into DNA. [3H]FdUrd was anabolized exclusively to FdUrd monophosphate, and preexposure to dFdCyd did not affect FdUrd monophosphate formation. Thymidylate synthase catalytic activity was inhibited by 48% after a 4-h exposure to 10 nM FdUrd and by 80% after exposure to the combination. Sequential 4-h exposures to 15 nM dFdCyd and 10 nM FdUrd led to greater depletion of dTTP pools (29% of control) than with either drug alone. Greater effects on nascent DNA integrity were seen with sequential dFdCyd followed by FdUrd. Although parental DNA damage was not evident immediately after exposure to 15 nM dFdCyd for 4 h followed by 10 nM FdUrd for 24 h, high molecular mass DNA fragmentation was evident 72-96 h after drug removal. Sequential dFdCyd/FdUrd was associated with prominent disturbance of the cell cycle, dTTP pool depletion, dATP/dTTP imbalance, and nascent DNA damage. Induction of double-strand parental DNA damage and cell death was delayed, consistent with postmitotic apoptosis.

Original languageEnglish (US)
Pages (from-to)2811-2818
Number of pages8
JournalClinical Cancer Research
Volume4
Issue number11
StatePublished - Nov 1 1998

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gemcitabine
Deoxyuridine
DNA Fragmentation
Colonic Neoplasms
DNA Damage
Cell Cycle
5-fluoro-2'-deoxyuridine
Pharmaceutical Preparations
Antimetabolites
Thymidylate Synthase
DNA

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Cytotoxicity and DNA fragmentation associated with sequential gemcitabine and 5-fluoro-2'-deoxyuridine in HT-29 colon cancer cells. / Ren, Qianfang; Kao, Vivian; Grem, Jean L.

In: Clinical Cancer Research, Vol. 4, No. 11, 01.11.1998, p. 2811-2818.

Research output: Contribution to journalArticle

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abstract = "The combined cytotoxic effects of the antimetabolites gemcitabine (dFdCyd) and 5-fluoro-2'-deoxyuridine (FdUrd) were studied. Cytotoxicity, biochemical perturbations, and DNA damage seen with dFdCyd and FdUrd alone and in combination were evaluated in HT-29 human colon cancer cells. A 4-h exposure to dFdCyd followed by FdUrd for 24 h produced more than additive cytotoxicity and marked S-phase accumulation. Cells progressed through the cell cycle, however, after a 22-h drug-free interval. [3H]dFdCyd was rapidly metabolized to the 5'-triphosphate and incorporated into DNA. [3H]FdUrd was anabolized exclusively to FdUrd monophosphate, and preexposure to dFdCyd did not affect FdUrd monophosphate formation. Thymidylate synthase catalytic activity was inhibited by 48{\%} after a 4-h exposure to 10 nM FdUrd and by 80{\%} after exposure to the combination. Sequential 4-h exposures to 15 nM dFdCyd and 10 nM FdUrd led to greater depletion of dTTP pools (29{\%} of control) than with either drug alone. Greater effects on nascent DNA integrity were seen with sequential dFdCyd followed by FdUrd. Although parental DNA damage was not evident immediately after exposure to 15 nM dFdCyd for 4 h followed by 10 nM FdUrd for 24 h, high molecular mass DNA fragmentation was evident 72-96 h after drug removal. Sequential dFdCyd/FdUrd was associated with prominent disturbance of the cell cycle, dTTP pool depletion, dATP/dTTP imbalance, and nascent DNA damage. Induction of double-strand parental DNA damage and cell death was delayed, consistent with postmitotic apoptosis.",
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