Investigating the role of DNA double strand break repair in determining sensitivity to radiotherapy fraction size

The dose of curative radiotherapy (RT) for cancer is commonly limited by adverse effects presenting years later. Late reacting normal tissues are, on average, more sensitive to the size of daily doses (fractions) than early reacting normal tissues and cancers. Clinical trials have shown breast cancers to be one exception to this rule, in that they are as sensitive to fraction size as the late reacting normal tissues. This has led to the adoption of hypofractionation (use of fractions >2.0 Gy) in the UK for the adjuvant therapy of women with early breast cancer. An understanding of the molecular basis of fraction size sensitivity is necessary to improve radiotherapy outcome. In this respect, it is relevant that late reacting normal tissues have lower proliferative indices than early reacting normal tissues and most cancers. Here, we test the hypothesis that tissue sensitivity to fraction size is determined by the DNA repair systems activated in response to DNA double strand breaks (DSB), and that these systems vary according to the proliferative status of the tissue.
Clinical data suggest that sensitivity of epidermis to fraction size varies over a 5-week course of RT. It resembles a late reacting normal tissue in its sensitivity to fraction size in the first week of RT and loses fractionation sensitivity by weeks 4 & 5. We used this feature of human epidermis to test how fractionation sensitivity and DNA repair changed over 5 weeks of RT. Breast skin biopsies were collected 2 h after the 1st, 5th and last fractions from 30 breast cancer patients prescribed 50 Gy/25fractions/5weeks. Sections of epidermis were co-stained for Ki67, cyclin A, p21, RAD51, 53BP1 and β1-Integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per mm of basement membrane (p=0.002), of which the majority were in S/G2 phase as judged by cyclin A staining (p<0.0003). The p21 index rose from 2.8% to 87.4% (p<0.0001) after 25 fractions, indicating cell cycle arrest in the basal epidermis. By week 5, there was a 4-fold increase (p=0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, confirming an association between activation of homologous recombination (HR) and loss of tissue fractionation sensitivity.
Subsequently, CHO cell lines deficient in specific DNA repair genes were used to test molecular pathways involved in sensitivity to fraction size. We irradiated AA8 (WT), irs-1SF (XRCC3-), V3-3 (DNA-PK-) and EM9 (XRCC1-) with 16 Gy gamma-rays in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA double-strand break (DSB) repair kinetics (RAD51 & 53BP1 staining) and cell cycle analysis using flow cytometry. We found that wild-type and DNA repair defective cells acquire resistance to fractionated radiotherapy by accumulation in the late S/G2 phase of the cell cycle and increased use of HR. In contrast, the irs1SF cells, defective in HR, failed to acquire radioresistance and remained equally sensitive to ionizing radiation throughout the 3-week treatment. We also demonstrated that sensitivity to fraction size is associated with functional NHEJ. It was undetectable in V3-3 cells lacking NHEJ and thereby likely relying on HR. The high fidelity of HR, which is independent of induced DNA damage levels and hence, of fraction size, may explain the low fractionation sensitivity of cells using HR to repair radiation induced DSBs.
We then wanted to investigate the modifying effects of small molecule inhibitors of DNA repair on fractionation responses. To this end we tested the effects of adding selected ATM, PARP, and DNAPK inhibitors to fractionated radiotherapy in WT CHO cells. Our results showed that the ATM inhibitor had a significant radiosensitising effect when combined with fractionated RT and resulted in loss of sparing effect of fractionation in wild type CHO cells, an observation that may be clinically relevant. We also examined DNA DSB repair kinetics (RAD51 & 53BP1 foci) with these drugs in the context of fractionated IR.

Keywords: cell cycle
DNA double-strand break repair
fraction size sensitivity
radiosensitisers
DNA repair inhibitors
fractionated radiotherapy
Advisor: Helleday, T
Yarnold, J