Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death.

A Yoshioka,S Tanaka,O Hiraoka,Y Koyama,Y Hirota,D Ayusawa,T Seno,C Garrett,Y Wataya

doi:10.1016/s0021-9258(18)47554-0

Abstract

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.

Highlights

It is known us to propose that DNA double strand breaks play an that thesize of intracellular deoxyribonucleoside triphosphate important role in the mechanism of FdUrd-mediated pools is 1000-fold smaller than the total amount of cell death
A comparison of the ratio of single and deoxyribonucleotideresidues inDNA [9]. It is alsoknown double strand breaks induced by FdUrd to that ob- that the dNTPpool imbalance can have profound effects on served following radiation suggested that FdUrd pro- the accuracy of DNA replication in cultured mammaliancells duced double strandbreaks exclusively
Our results indicate that imbalance of the dNTPpools is a trigger for double strand DNA break andcell death

Summary

EXPERIMENTAL PROCEDURES AND RESULTS*

Anisms of action of these compounds are still not entirely clear. 5-FU and 5-fluorodeoxyuridine (FdUrd) are converted to 5-fluorodeoxyuridine 5‘-phosphate, which is a potent in-. The DNA double strand breaksinduced by dNTP the dGTP pool decreased to a level below the limits of the imbalance were not repaired, and boththe DNA breakage and measurement, and after h agreat imbalance of dNTP pools the cell death induced by FdUrd were prevented by addition was observed; the dATP and dCTPpools increased to about of cycloheximide. Were assayed for double strand endonuclease activity both In order to elucidate what kind of dNTP pool imbalance before and 18 hrafter exposure to FdUrd. The enzymic causes the cell death, we carried out experiments in which activity was assayed by incubating [14C]-labeled FM3A DNA deoxyadenosine, deoxyguanosine, deoxycytidine, or deoxywith aliquots of cell lysate, followed by neutral filter elution thymidine was added to FM3A cultures and the dNTP pool analysis for the DNA breakage. 90 "C for 30 min or when i t was treated with trypsin

DISCUSSION

RESULTS

Determlnatlon of strandbreakiaqe of parental DNA I"