Format

Send to

Choose Destination
Int J Radiat Oncol Biol Phys. 1994 Jul 30;29(5):1035-40.

The repair fidelity of restriction enzyme-induced double strand breaks in plasmid DNA correlates with radioresistance in human tumor cell lines.

Author information

1
Radiation Research Unit, Institute of Cancer Research, Sutton, Surrey, UK.

Abstract

PURPOSE:

The accuracy of DNA repair may play a role in determining the cytotoxic effect of ionizing radiation. Repair, as measured by DNA strand breakage, often shows little difference between tumor cell lines of widely different radiosensitivity. The mechanism by which DNA fragments are rejoined is poorly understood. This study used plasmid transfection as a probe to assess the balance between correct repair and misrepair.

METHODS AND MATERIALS:

Using techniques described, a double-strand break was introduced into a coding sequence of circular plasmid DNA using a restriction endonuclease as a model for a radiation-induced double-strand break; it was then transfected as a linear molecule into human tumor cells, and the subsequent cell-mediated restoration of the coding sequence, evidenced by intact gene function, was documented. The plasmid used in these experiments, pPMH16, is known to integrate into genomic DNA. Gene function was tested by the ability to grow colonies in selection media. The plasmid also contains a second selectable marker gene that was used to identify transfected cells, before the function of the damaged gene was tested. The proportion of transfected cells that had correctly restored the damaged gene gave a measure of repair fidelity.

RESULTS:

A general trend for sensitive cells to show lower repair fidelity relative to resistant cells was observed. The type of double-strand cleavage of the plasmid (staggered or blunt) made little difference to the measured repair fidelity, in contrast to published studies in which restriction-enzyme breaks had been introduced into DNA within chromatin. Specific comparison of parent lines and their radiosensitive clones showed significant differences in repair fidelity for a relatively small change in radiation response, which was in line with the overall correlation. These same pairs have previously been shown to have no difference in the loss of DNA fragmentation with time after irradiation, and Southern analysis had confirmed the integrated plasmid copy number was similar in the cell lines compared. The number of intact copies of the damaged gene relative to the undamaged gene mirrored the observed repair fidelity. However, in one cell line out of the 10 studied, an exception to the observed trend was found. In a comparison of two equally radioresistant bladder cancer cell lines, large differences in repair fidelity were observed. Again, no difference in the integrated copy number was found, and the damaged gene was highly rearranged or deleted in the cell line with low repair fidelity.

CONCLUSION:

These studies have shown repair fidelity to correlate closely with radiosensitivity, including the comparison of genetically related lines. It is suggested that repair fidelity can be, but is not invariably, a measure of correct repair relative to misrepair, resulting from the processing of double-strand breaks and, hence, the response to ionizing radiation.

PMID:
8083072
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center