B. Marples, P. Johnston, S.D. Scott, G.D. Wilson and M.C. Joiner
Gray Laboratory CRT, Mount Vernon Hospital, Northwood, Middlesex, HA6
2JR, UK
Over the last decade we have demonstrated that radiation sensitivity can be dose-dependent with small acute exposures (typically below ~50 cGy) being more lethal per unit dose than larger exposures (> 1 Gy). This change in radiosensitivity has given rise to the descriptive terms low dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR). HRS may be the constitutive response to low-dose radiation exposures of all normal and malignant cell systems (both in vivo and in vitro) since it is found in ~80% of cell lines we have investigated. Evidence suggests that IRR reflects an induced 'protective' repair response triggered by accumulated DNA damage, as evidenced by our dose-rate experiments.
We are investigating the underlying mechanism of HRS/IRR. In a panel of 10 cell lines of differing HRS/IRR response, we have established that DNA-PK activity, and not the amount of DNA-PK present, may dictate the IRR response. This observation is supported by work using the MO59 family of cell lines that express disparate DNA-PK status. MO59J cells, that lack DNA-PK, did not show an IRR response. In contrast, MO59K and MO59J/FUS1 cells that contain DNA-PK both exhibited distinct IRR responses. The HRS response was indistinguishable in the three MO59 cell lines.
The specific poly (ADP-ribosyl) transferase (PARP) inhibitor NU1025 prevented the development of IRR. In contrast, NU1025 did not affect the survival response in U373 cells that lack an HRS/IRR response. These data confirm our earlier observations with the less specific PARP inhibitor 3-aminobenzamide. It is likely therefore that DNA damage detection via a PARP-mediated process is involved in the initiation of the IRR response. To test this hypothesis, we have recently generated a functional PARP specific ribozyme.
The involvement of DNA strand breaks in HRS/IRR has recently been confirmed with our preliminary observations that HRS/IRR can be detected using the micronuclei assay. Of the cell lines we have investigated to date, a correspondence exists between the presence of HRS/IRR scored by clonogenic survival or the micronuclei assay. Our in-house developed high-resolution comet assay is being used to measure DNA strand breaks in the dose region characterising HRS/IRR. This has required modifications and improvements to both cell lysis protocols and comet scoring algorithms. The measurement of DNA damage after 0-2 Gy doses is currently in progress.
While the precise mechanism underlying HRS/IRR has yet to be fully determined the balance of evidence is now indicating that DNA repair activity involving PARP signalling and DNA-PK transduction is critical.