Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

Abstract

Abbreviations used: BrdUrd -bromodeoxyuridine; Cs -cesium; DAPI -4',6-diamidino-2-phenylindole; dp -delayed plating; DSB -double-strand breaks; FACS -fluorescent activated cell sorter; FCS -fetal bovine calfserum; HEPES -2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HPV -human papilloma virus; ip -immediate plating; PBSphosphate buffered saline; PLD -potentially lethal damage; PLDR -potentially lethal damage repair; PVDF -polyvinylidene fluoride; RIPA -radio immunoprecipitation assay; SDS PAGE -sodium dodecyl sulfate polyacrylamide gel electrophoresis; TNBS -PBS containing 0.1% Triton X-100 and 1% FCS Abstract:The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD 2 ). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a 137 Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Analysis of 177Lu-DOTA-Octreotate Therapy–Induced DNA Damage in Peripheral Blood Lymphocytes of Patients with Neuroendocrine Tumors

“…The absorbed dose to bone marrow can affect the genomic stability of progenitor cells and their progeny. Individual radiation sensitivity influences large variability in clinical response to radiotherapy, as well as cellular radiosensitivity measured by the g-H2AX assay in PBLs (7,14,15). There is also an ongoing debate on the mechanisms of the radiation-induced bystander effect (25), which may contribute to an elevated appearance of dose to circulating lymphocytes through reactive oxygen and nitrogen species and immune response.…”

“…The direct response to radiation and the sensitivity of the assay have provided a basis for the adoption of g-H2AX as a "biodosimeter" to evaluate exposure of peripheral blood lymphocytes (PBLs) after external body irradiation (9)(10)(11)(12)(13). Recent publications evaluated the g-H2AX assay as an attractive functional assay for identification of radiosensitive individuals with early or chronic radiation-induced normal-tissue toxicity and for personalized therapy (7,14,15).…”

Effect of X-ray minibeam radiation therapy on clonogenic survival of glioma cells

“…Although our findings are contradicting the dogma of existing PLDR capacity in most mammalian cells, cell lines without PLDR have been reported previously [20] . The mutant p53 status of the F98 cell line [21] together with its very low expression level [22] , might be an explanation for the absence of PLDR as PLD expression has been suggested to be primarily dependent on p53 [23] , [24] . In addition to impaired p53 function, they exhibit mutant p16/CDKn2y/INK4 and a negative BRCA1 localization after X-rays [25] , which could further contribute to the absence of PLDR in these cells.…”

Targeting histone deacetylase 4/ubiquitin‐conjugating enzyme 9 impairs DNA repair for radiosensitization of hepatocellular carcinoma cells in mice

“…The reduction of Rad51/ϒH2AX foci observed in irradiated HCC cells after panobinostat pretreatment likely indicates that panobinostat inhibits Rad51 recruitment to DSBs and thereby reduces DNA repair. Panobinostat pretreatment resulted in the persistence of ϒH2AX foci at 24 hours after irradiation in HCC cells, which was thought to reflect the lethality of the DNA damage . Many proteins, in addition to Rad51 and 53BP1, have been shown to form nuclear foci following DNA damage, including ataxia telangiectasia mutated, breast cancer 1, and MRE11/RAD50/NBS1 complex .…”

“…Panobinostat pretreatment resulted in the persistence of !H2AX foci at 24 hours after irradiation in HCC cells, which was thought to reflect the lethality of the DNA damage. (32,33) Many proteins, in addition to Rad51 and 53BP1, have been shown to form nuclear foci following DNA damage, including ataxia telangiectasia mutated, breast cancer 1, and MRE11/RAD50/NBS1 complex. (34)(35)(36) HDAC inhibitors have also been reported to decrease the amount of Rad51 protein and inhibit HR repair in response to irradiation.…”