Molecular Pharmacology, Vol 2, 1-9, Copyright © 1966 by the American Society for Pharmacology and Experimental Therapeutics

DNA Synthesis by Mammalian Cells Inhibited in Culture by 5-Iodo-2'-deoxyuridine

¹ Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut

In the presence of 5-iodo-2'-deoxyuridine (IUdR; 1-2 x l0^-4 M) P815Y murine mast tumor cells in culture complete only one doubling of their cell number and one doubling of their DNA. Even though DNA does not increase beyond this one doubling, DNA synthesis continues in IUdR-inhibited cultures. ³H-Thymidine is incorporated into DNA, and the pattern of the incorporation as seen with CsCl density gradient centrifugation agrees with the pattern that would be expected from replication of hybrid DNA containing IUdR in one strand (IU-THY DNA) in medium without IUdR. A new band appears at the density of native DNA, and both the new band and the IU-THY band are radioactive. Because in all experiments the specific radioactivity of the IU-THY band was about one-half that of the native DNA band, it is suggested that only one-half of the IU-THY DNA replicates.

The failure of the amount of DNA in the culture to increase despite the continued synthesis of DNA suggested that breakdown of DNA must occur in IUdR-inhibited cultures. IU-THY DNA was labeled with radioactivity in either the IUdR-containing strand or in the thymidine-containing strand. Both strands were found to break down at about the same rate, and the rate of breakdown appeared to be sufficient to account for the failure of DNA to accumulate in the inhibited culture. This experiment also indicated that the breakdown of DNA was not a consequence of DNA "repair," since the distinguishing feature of "repair" is selective removal of abnormal DNA.

Note:
ACKNOWLEDGMENTS The authors wish to thank Dr. Pauline Chang for the ³H-IUdR. We are indebted to Mrs. Agneta Brown, Miss Katja Hahn, and Mrs. Donna Reno for their very able technical assistance. This investigation was supported by Public Health Service Research grant No. CA-02817-09 from the National Cancer Institute. N. R. Morris is an American Cancer Society Scholar in Cancer Research; supported by grant No. P333 of the American Cancer Society.