Transcriptional responses to ionizing radiation reveal that p53R2 protects against radiation-induced mutagenesis in human lymphoblastoid cells

Abstract

The p53 protein has been implicated in multiple cellular responses related to DNA damage. Alterations in any of these cellular responses could be related to increased genomic instability. Our previous study has shown that mutations in p53 lead to hypermutability to ionizing radiation. To investigate further how p53 is involved in regulating mutational processes, we used 8K cDNA microarrays to compare the patterns of gene expression among three closely related human cell lines with different p53 status including TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNA samples were collected at 1, 3, 6, 9, and 24 h after 10 Gy gamma-irradiation. Template-based clustering analysis of the gene expression over the time course showed that 464 genes are either up or downregulated by at least twofold following radiation treatment. In addition, cluster analyses of gene expression profiles among these three cell lines revealed distinct patterns. In TK6, 165 genes were upregulated, while 36 genes were downregulated. In contrast, in WTK1 75 genes were upregulated and 12 genes were downregulated. In NH32, only 54 genes were upregulated. Furthermore, we found several genes associated with DNA repair namely p53R2, DDB2, XPC, PCNA, BTG2, and MSH2 that were highly induced in TK6 compared to WTK1 and NH32. p53R2, which is regulated by the tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in radiation-induced mutagenesis, p53R2 protein was inhibited by siRNA in TK6 cells and followed by 2 Gy radiation. The background mutation frequencies at the TK locus of siRNA-transfected TK6 cells were about three times higher than those seen in TK6 cells. The mutation frequencies of siRNA-transfected TK6 cells after 2 Gy radiation were significantly higher than the irradiated TK6 cells without p53R2 knock down. These results indicate that p53R2 was induced by p53 protein and is involved in protecting against radiation-induced mutagenesis.

Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis

“…Given that RR subunits are a likely target of ATM signaling (14,20) and considering our observation that the mtDNA copy number increase in response to IR was RR dependent, we examined the relative expression of the R1, R2, and p53R2 subunits of RR after IR by Western blotting. As reported by others (18,20,(51)(52)(53), we observed an increase in p53R2 and a marked reduction of R2 in wild-type fibroblasts exposed to IR ( Figure 2D). In addition, there was a slight reduction in the amount of the R1 subunit.…”

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

“…S5 and S6). Concordant with the idea that CycG2 is upregulated in response to activation of DNA DSB signaling pathways, previous studies indicated significant elevation of CCNG2 mRNAs upon ␥-irradiation-induced DNA damage (64). Because the doxorubicin-stimulated increase in CycG2 levels is clearly detectable within 4 h of dosing, well before an obvious arrest at the G 2 /M boundary ( Fig.…”

Gene Expression Profiling of Breast, Prostate, and Glioma Cells following Single versus Fractionated Doses of Radiation

“…Methods for RNA extraction, probe labeling reactions, and microarray hybridization were as described previously (8). The microarray slides used for the experiments contained 7,680 human cDNA clones, and the details of this array and the methods for microarray fabrication were described previously (9).…”