Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

Abstract

The topoisomerase IIalpha inhibitor etoposide is a 'broad spectrum' anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK(cs) as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G(2)/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G(2)/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G(2)/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

“…Etoposide is a highly active inducer of DNA double strand breaks and G 2 arrest in mammalian cells (14,15). No G 2 /M arrest was observed following the exposure of the two CCA cell lines to etoposide alone.…”

“…Etoposide induces DNA double-strand breaks during the S and G 2 phases of the cell cycle via inhibition of DNA topoisomerase II activity (15). It is highly likely that etoposide-induced DNA damage prior to irradiation renders cancer cells more vulnerable to the effects of radiation.…”

Role of the pro-survival molecule Bfl-1 in melanoma

“…Indeed, after Etoposide treatment, previous work has consistently shown cell cycle arrest in the G2/M phase, and the resulting translation to cell death, across a range of tumour types 466,469,470 . Etoposide forms a complex with topoisomerase II on DNA, so that cleaved DNA breaks cannot be repaired.…”

p53 Protein-mediated Up-regulation of MAP Kinase Phosphatase 3 (MKP-3) Contributes to the Establishment of the Cellular Senescent Phenotype through Dephosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2)

“…*, p Ͻ 0.05 versus respective untreated control. senescent model induced by ETO (17). Incubation of NRK cells with subtoxic concentrations of ETO resulted in an enlargement of cell size and elevation of SA-␤-gal activity, indicative of cellular senescence (Fig.…”