Elsevier

Biochemical Pharmacology

Volume 48, Issue 8, 18 October 1994, Pages 1619-1630
Biochemical Pharmacology

Molecular effects of 2′,2′-difluorodeoxycytidine (gemcitabine) on DNA replication in intact HL-60 cells

https://doi.org/10.1016/0006-2952(94)90207-0Get rights and content

Abstract

The ability of pH-step alkaline elution to isolate different size species of nascent DNA (nDNA) from intact cells was utilized to study the effects of 2′,2′-difluorodeoxycytidine (dFdC) on DNA replication in HL-60 cells. Preincubation with dFdC caused a concentration-dependent decrease in overall [3H]thymidine incorporation into DNA, accompanied by an increase in the proportion of radiolabel accumulated in small nDNA fragments. Twenty-four hours following removal of dFdC, radiolabel progressed from smaller to larger fragments and into genomic-length DNA. At initial concentrations of exposures to dFdC or cytosine arabinoside (ara-C) that caused 50% lethality (lc50) to HL-60 cells (40 and 50 nM, respectively), slower and less complete transit of nDNA from small subreplicon-length fragments through larger intermediates to genomic-length DNA was observed for nDNA fragments containing incorporated [3H]dFdC than for fragments containing [3H]ara-C. This was accomplished with less [3H]dFdC incorporated into DNA than [3H]ara-C at these extracellular concentrations of drug. Pulse-chase studies, using higher concentrations of radiolabeled drug, similarly revealed that nDNA fragments containing incorporated dFdC, like those containing ara-C, progressed with respect to time into larger nDNA intermediates and ultimately into genomic-length DNA; however, such progression for nDNA fragments containing dFdC was less complete than for fragments containing ara-C. The radioactivity incorporated into DNA represented authentic dFdC, as determined by DNA degradation studies, and was stable in DNA for at least 48 hr after removal of extracellular [3H]dFdC. Some of the effects of dFdC on ribonucleotide reduction in HL-60 cells were assessed by measurement of the intracellular pools of dCTP and dGTP. The drug had a greater effect on pools of dGTP than of dCTP, with transient reductions in dGTP observed at concentrations that encompass the lc50 for dFdC. These studies suggest that the interaction with DNA synthesis is an important component of the cytotoxicity of dFdC in HL-60 cells. Because it is incorporated progressively through nDNA compartments and ultimately into genomic-length DNA, dFdC should be categorized as an agent that slows DNA elongation in the intact cell, and not as a chain terminator in the absolute sense.

References (47)

  • JL Abbruzzese et al.

    A Phase I clinical, plasma, and cellular pharmacology study of Gemcitabine

    J Clin Oncol

    (1991)
  • V Fink et al.

    Phase II study of Gemcitabine in metastatic colorectal cancer

  • H Anderson et al.

    Phase II study of Gemcitabine in non-small cell lung cancer (NSCLC)

  • R Abratt et al.

    Efficacy and safety of Gemcitabine in non-small cell lung cancer

  • E Eisenhauer et al.

    Gemcitabine is active in patients with previously untreated extensive small cell lung cancer—A Phase II study of the National Cancer Institute of Canada Clinical Trials Group

  • M Clavel et al.

    Gemcitabine is an active drug in patients with squamous cell carcinoma of the head and neck

  • P de Mulder et al.

    Phase II study of Gemcitabine in renal cancer

  • CF Pollera et al.

    Weekly Gemcitabine: a Phase I study with short and prolonged infusion schedules

  • J Carmichael et al.

    Gemcitabine: An active drug in advanced breast cancer. Results of a Phase II study

  • V Ghandi et al.

    Modulatory activity of 2',2'-difluorodeoxycytidine on the phosphorylation and cytotoxicity of arabinosyl nucleosides

    Cancer Res

    (1990)
  • MJ Keating et al.

    Improved prospects for long-term survival in adults with acute myelogenous leukemia

    JAMA

    (1982)
  • V Heinemann et al.

    Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-β-d-arabinofuranosylcytosine

    Cancer Res

    (1988)
  • JJ Furth et al.

    Inhibition of mammalian DNA polymerase by the 5'-triphosphate of 1-β-d arabinofuranosylcytosine and the 5'-triphosphate of 9-β-d arabinofuranosyladenine

    Cancer Res

    (1968)
  • Cited by (42)

    • Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine

      2014, Journal of Biological Chemistry
      Citation Excerpt :

      Gemcitabine (2′,2′-difluoro-2′-deoxycytidine, dFdC; Fig. 1) is widely used in the treatment of various carcinomas, including pancreas, lung, breast, and bladder cancer (2). Phosphorylated dFdC metabolites act as inhibitors of ribonucleotide reductase and CTP synthetase, resulting in decreased intracellular dNTP (i.e. dCTP) levels, and the drug functions as a DNA chain terminator when incorporated as its 5′-triphosphate metabolite (dFdCTP) into the DNA (3, 4). First dFdC is phosphorylated to its 5′-monophosphate derivative (dFdCMP) by 2′-deoxycytidine kinase.

    • Antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo

      2017, Antiviral Research
      Citation Excerpt :

      HRV1B infection induced mild but distinct infiltration of inflammatory cells, mainly lymphoid cells and a few neutrophils, in the terminal air ways and alveoli, which was ameliorated by treatment with gemcitabine (Fig. 5D∼5F). Gemcitabine triphosphate (dFdCTP), the main metabolite of gemcitabine (dFdC) generated by deoxycytidine kinase, competes with dCTP for incorporation into DNA, and results in DNA damage (Heinemann et al., 1990; Ross and Cuddy, 1994). Gemcitabine inhibits DNA polymerase and other enzymes associated with deoxycytidine metabolism, including ribonucleotide reductase (Heinemann et al., 1990), deoxycytidylate deaminase (Heinemann et al., 1992), and CTP synthetase (Heinemann et al., 1995).

    • Monitoring genotoxicity in patients receiving chemotherapy for cancer: application of the PIG-A assay

      2016, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
      Citation Excerpt :

      Patients 07 and 26, both of whom had high PIG-A MFs, were treated with gemcitabine (GEM) and TS-1, respectively, in the absence of radiation therapies, suggesting that the higher PIG-A MFs were caused by chemotherapy but not by radiation therapy (Fig. 1 and Tables 1 and 2). GEM is a fluorinated deoxycytidine analogue that inhibits ribonucleotide reductase and can be incorporated into DNA, thus inhibiting DNA replication [21–23]. In mice, GEM treatment induced a significant increase in total chromosomal aberrations and the frequency of micronuclei in vivo [24].

    • Radiation modifiers: Treatment overview and future investigations

      2006, Hematology/Oncology Clinics of North America
    View all citing articles on Scopus

    Presented in part at the 83rd Annual Meeting of the American Association for Cancer Research, Orlando, FL, May 1993, and to the Workshop on Gemcitabine: Status of Pre-Clinical Studies, Free University Hospital, Amsterdam, The Netherlands, March 1994.

    View full text