TP53 modulates radiotherapy fraction size sensitivity in normal and malignant cells

Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential to fully exploit this therapeutic variable for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is dependent on the presence of wild-type (WT) p53 and intact non-homologous end-joining (NHEJ). Using single or split-doses of radiation in a range of normal and malignant cells, split-dose recovery was determined using colony-survival assays. Both normal and tumour cells with WT p53 demonstrated significant split-dose recovery, whereas Li-Fraumeni fibroblasts and tumour cells with defective G1/S checkpoint had a large S/G2 component and lost the sparing effect of smaller fractions. There was lack of split-dose recovery in NHEJ-deficient cells and DNA-PKcs inhibitor increased sensitivity to split-doses in glioma cells. Furthermore, siRNA knockdown of p53 in fibroblasts reduced split-dose recovery. In summary, cells defective in p53 are less sensitive to radiotherapy fraction size and lack of split-dose recovery in DNA ligase IV and DNA-PKcs mutant cells suggests the dependence of fraction size sensitivity on intact NHEJ

Table S1 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

List of primers and antibodies.</p

Table S3 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

A) Assessment of acute large bowel damage. CD1-nude mice treated with mock treatment or PAN (10 mg/kg) +/-IR. Lesions were scored semi-quantitatively using a 0-5 scale where 0 is no lesion present to 5 where the entire organ was affected by the pathology. B) Assessment of damage to intestine and bladder at 12 weeks. P=lesion present; scoring is semi-quantitative. Cage 2 mouse 1 was culled 2 days after being irradiated in error without the collimator present and cage 2 mouse 5 became unwell 7.5 weeks post-treatment and was culled, but no histopathological effects of the treatment were observed in the bowel. Cage 4 mouse 4 developed moderate diffuse ulcerative colitis. Amyloid was present in the small intestine of animals from all groups including the control group and is considered a pre-existing spontaneous lesion not related to the experimental protocol. Lesions were scored semi-quantitatively using a 0-5 scale where 0 is no lesion present to 5 where the entire organ was affected by the pathology.</p

Figure S3 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

Radiosensitising effects of panobinostat under hypoxia. A) Effect of 24-hour 25 nM PAN or DMSO on radiosensitivity of RT112 cells under 2% O2 (hypoxia) and normoxia and exposure to irradiation under the same conditions. Normoxia data taken from (14).</p

Figure S4 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

HDAC expression and cellular response to HDAC inhibition. A) qPCR of mRNA expression of HDAC 1-11 genes in normal human urothelial cells (NHU) and RT112, CAL29 and T24 bladder cancer cells. Relative gene expression was normalised to GAPDH levels (n=3); B) Effects of panobinostat on expression of selected HDAC proteins (n=2); C) Clonogenic assay representing cytotoxicity of mocetinostat in RT112, CAL29 and T24 bladder cancer cells, after 24 h incubation (n=3); D) Clonogenic assay representing cytotoxicity of TMP195 in RT112 cells, after 24 h incubation (n=3); E) Effect of panobinostat on acetylation levels of histone H3 lysine 18 (H3K18) at 0.5, 1, 2, 3 and 24 h incubation in RT112 cells (n=2); F) Quantification of western blots in Figure 6 (n=3 for MRE11, HDAC1 and NBS1, n=2 for HDAC2 and RAD51); G) HR assay results for TMP195 (n=3).</p

Table S2 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

Panobinostat levels following IP and IV administration. Six h after injection of PAN 10 mg/kg IP or IV, plasma and selected organs were removed and PAN concentrations determined using HPLC.</p

Figure S1 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

Days for RT112 xenografts to treble in volume and body weights and treatment of Ku80KD xenografts. A) Days for xenografts to treble in volume (PAN+IR vs IR, p=0.06). Skin overlying the xenograft developed ulceration in two mice treated with IR alone and four mice in the PAN+IR group, beyond the time to treble tumour volume; B) Body weight in mice carrying RT112 xenografts, vehicle (n=5), PAN (n=7), PAN+IR (n=6) or IR alone (n=6); C) Mouse body weights in mice carrying 30% KuKD xenografts for vehicle (n=4), PAN (n=6), PAN+IR (n=6) or IR alone (n=6); D) Ku80 knock-down cells express approximately 70% protein; E) Treatment of Ku80KD xenograft mice.</p

Supplementary methods from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

Supplementary methods.</p

Figure S2 from Radiosensitization <i>In Vivo</i> by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity

Treating mice vertically head-down in the SARRP. Preliminary experiments on freshly dead mice, irradiated then immediately subjected to autopsy, demonstrated that small bowel is at a safe distance from the field edge.</p