Search

The rejoining of radiation-induced DNA breaks, characterized by 5′ termini, was measured by the polynucleotide kinase method. This method has potential clinical value because DNA damage can be measured for cells within solid tissue. Following irradiation of a human squamous-cell carcinoma, DNA rejoining occurred, but at a slower rate than observed for mammalian cells in culture. The results of the study indicate that cells within solid tumors, in situ, are able to repair at least some radiation-induced DNA damage. The importance of this type of repair to tumor response following therapeutic radiology is not known at present.

L cells irradiated while in growth medium or in a balanced salts solution showed a rapid appearance of DNA breaks characterized by 5′ phosphoryl termini. Following irradiation, the number of breaks quickly reached a maximum, after which they were rapidly repaired. Dinitrophenol, an inhibitor of aerobic oxidative phosphorylation, prevented the DNA breaks from appearing.

The production and rejoining of X-ray-induced single-stranded DNA breaks was studied using the alkaline sucrose density gradient technique and by measuring the disappearance of both 5' termini and 3'-OH termini using polynucleotide kinase and DNA polymerase, respectively. All studies were conducted using L-cell suspensions irradiated both in the presence and absence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. Results show that the induction of single-stranded DNA breaks probably includes a nucleolytic component in addition to indirect free radical effects. A greater number of breaks were produced in the absence of DNP, suggesting that depressed adenosine triphosphate (ATP) levels reduce endogenous nucleolytic activity. The rejoining mechanism is enzymatic and requires an available ATP supply for operation. In the presence of DNP no DNA rejoining was observed following 30 min incubation after 10,000 rad. These results suggest that DNA breaks produced may be characterized by 5'-PO(4)-3'-OH termini and are rejoined by DNA ligase.

Using the polynucleotide kinase method, we were able to demonstrate the repair of radiation induced DNA breaks in mouse liver DNA and in L cells. The specificity of the enzyme indicates the breaks to have 5′ phosphoryl termini.

In an earlier paper we attempted to show the “cut-and-patch” mechanism to be responsible for the repair of radiation induced DNA damage in cultured mammalian cells. The experimental basis was provided by survival studies. For the present investigation we used the polynucleotide kinase method to measure directly the appearance and rejoining of radiation induced DNA breaks characterized by 5′ PO 4 termini. A multicompartmental mathematical model was developed, and estimates of parameter values were made by experiments with L cells. The results suggest most of the frank DNA breaks following irradiation to be a consequence of endogenous nucleolytic processes and not due to the radiation, per se . Because of the massive cell killing which occurs in spite of preventing the appearance of DNA breaks, we postulate the existence of radiation induced DNA changes which (1) are not liable to endogenous nucleolytic processes and (2) cause cell lethality.

Experiments with bacteria suggest that extensive DNA degradation is an important step in the repair of radiation injury. Because of the potential importance, similar experiments with cultured mammalian cells were performed. L cells prelabeled with ${}^{3}{\rm HTdR}$ were suspended in medium and irradiated with doses of 0, 100, 500, and 1000 rads of 250 kVp x-rays. The results showed: (a) No massive DNA degradation occurred during the first 6 postirradiation hours; (b) although a small amount of radioactivity is released into the suspending medium, this is not increased by increasing dose of radiation; (c) the release of the radioactivity occurred in the presence of 2,4-dinitrophenol-an uncoupler of aerobic oxidative phosphorylation; (d) most of the released radioactivity was in the form of thymidine; and (d) the intracellular concentrations of alkaline deoxyribonuclease and DNA polymerase were not increased after irradiation.

The uncoupler of aerobic oxidative phosphorylation, 2,4-dinitrophenol (DNP), has been previously shown to decrease the radiation sensitivity of L cells. In the present investigation we performed paired irradiation experiments with DNP-treated cells. Although DNP treatment virtually stops macromolecular synthesis, the paired-dose survival curve showed a definite "repair" segment. The absolute survival reached by the DNP-treated cells was greater than that in parallel irradiated control cells kept in complete growth medium.

Mouse L cells show a cyclic32 P labeling of DNA following relatively low doses of x-rays. This effect appeared for cells which had been starved or treated with 2,4-dinitrophenol (DNP) but not for cells irradiated in growth medium. The response consisted of a rapid uptake and loss of label. Changes in ATP metabolism paralleled the cyclic labeling of DNA. This behavior possibly explains the enhanced repair of radiation injury previously described for DNP treated cells.