Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells

Abstract

The relationship between cell cycle progression and induction of DNA double-strand breaks and cytotoxicity by exposure to fluorodeoxyuridine (FdUrd) was studied in HT29 human colon cancer cells. Fractionation of drug-treated populations by centrifugal elutriation yielded subpopulations having widely divergent abilities to progress through S phase in the presence of the drug. One of these subpopulations, which appeared to undergo coordinated growth arrest, was resistant to FdUrd cytotoxicity and DNA damage. In contrast, the subpopulation which was able to progress furthest through S phase in the presence of FdUrd underwent unbalanced growth arrest (i.e., increase in size and mass out of proportion to DNA synthesis), and displayed both DNA double-strand break formation (assayed by pulsed field gel electrophoresis) and loss of clonogenicity. When cells were elutriated prior to drug treatment, producing fractions enriched in cells at various cell cycle stages, no significant differences in sensitivity to FdUrd-induced cytotoxicity were detected among elutriation fractions. These findings support the model that, in HT29 cells, progression into and through S phase during drug treatment is an important determinant of FdUrd-induced DNA damage and cytotoxicity, but that the cell cycle position at the start of drug exposure is not a critical factor for these effects.

Fragmentation of Replicating Chromosomes Triggered by Uracil in DNA

“…Replication fork collapse has been proposed before as the mechanism behind double-strand DNA breaks caused by uracil misincorporation and subsequent excision. 35,46 We would like to extend this argument and propose that replication fork collapse is the process behind chromosomal fragmentation after any type of DNA damage, as long as the DNA lesions are repaired via excision. In this respect, replication fork collapse is likely to be the major source of chromosomal fragmentation in the wildtype cells, growing under a variety of conditions.…”

Identification of DNA Repair Pathways That Affect the Survival of Ovarian Cancer Cells Treated with a Poly(ADP-Ribose) Polymerase Inhibitor in a Novel Drug Combination

“…However, it is not known whether these lesions are substrates for other repair pathways that could productively repair them. Nor is it known whether attempted repair and/or replication of these lesions creates DNA damage that is more toxic, a possibility, considering that FdUrd induces double-strand DNA breaks (Yoshioka et al, 1987;Tang et al, 1996;Meyers et al, 2001). Finally, it is not clear whether PARP inhibition, which disrupts DNA repair, alters the lesions that are ultimately induced by FdUrd.…”

“…Our results also demonstrate that these repair pathways do not make lesions induced by FdUrd alone or FϩA more toxic. Given that FdUrd activates ATM and causes DSBs (Yoshioka et al, 1987;Tang et al, 1996;Meyers et al, 2001;Parsels et al, 2004;Wilsker and Bunz, 2007;Liu et al, 2008;Jardim et al, 2009;Geng et al, 2011; and given that FϩA induces more H2AX phosphorylation than FdUrd alone (see Fig. 1B), we were surprised that depletion of ATM or KU80, two proteins that participate in signaling from and repair of DSBs, did not affect the survival of cells treated with these agents.…”

5-Fluoro-2′-Deoxyuridine-Induced cdc25A Accumulation Correlates with Premature Mitotic Entry and Clonogenic Death in Human Colon Cancer Cells

“…Responses that involve regulation of cell cycle progression and apoptosis are particularly important in this regard (5,6,21). In previous studies in which we compared the effects of FdUrd in two human colorectal tumor cell lines (HT29 and SW620), we found that the propensity of HT29 cells to try to advance through the cell cycle during profound thymidylate synthase inhibition correlated closely with their sensitivity to FdUrd-induced clonogenic death, relative to SW620 cells (22)(23)(24). Based on these observations, we hypothesized that the sensitivity of HT29 cells to thymidylate synthase inhibition may be due in part to a defect in their cell cycle checkpoint response(s) to FdUrd-induced DNA damage.…”