Loss of wild-type Trp53 protein in mouse fibroblasts leads to increased radioresistance with consequent decrease in repair of potentially lethal damage.
It has been reported that the loss of function of Trp53 protein is associated with a reduction in the expression of radiation-induced potentially lethal damage (PLD). These studies, however, were carried out using either transformed or transfected cell lines, and other factors may have existed that could interfere with PLD repair. In this study, we used isogenic fibroblasts derived from Trp53 knockout mice to study radiation sensitivity, PLD repair, and repair of DNA double-strand breaks (DSBs). Experiments were carried out using wild-type (Trp53(+/+)), heterozygous (Trp53(+/-)) and homozygous mutant (Trp53(-/-)) cells. This is an ideal system because the only difference in the three cell strains is the status of the Trp53 protein. DSB repair was measured by pulsed-field-gel electrophoresis (PFGE), while radiosensitivity and PLD repair were studied using the clonogenic survival assay. Cells were irradiated in plateau phase and then trypsinized and plated either immediately or 24 h later to allow for PLD repair. The results of Western blot analyses showed that Trp53(-/-) cells expressed a putative mutant form of Trp53 that was unable to transcriptionally activate Cdkn1a (p21) protein in response to irradiation. The Trp53(-/-) cells were significantly more radioresistant than the Trp53(+/+) cells, and this was associated with a moderate reduction in PLD repair. DNA repair experiments showed no difference in DSB rejoining capability between the two cell lines. In conclusion, our results show that loss of wild-type Trp53 leads to increased radioresistance with consequent reduction in PLD repair but with no effect on DNA DSB repair.
Alsbeih
G
G
Biomedical Physics Department, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia. galsbeih@kfshrc.edu.sa
Torres
M
M
Al-Harbi
N
N
Alsubael
M
M
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D005786:Gene Expression Regulation / Q000528:radiation effects*; D051379:Mice; D011836:Radiation Tolerance / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53