Transcriptional changes of mitochondrial genes in irradiated cells proficient or deficient in p53 (original) (raw)
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Irradiation-induced Translocation of p53 to Mitochondria in the Absence of Apoptosis
Journal of Biological Chemistry, 2005
The tumor suppressor protein p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. Recently, it was shown that during apoptosis p53 can specifically translocate to mitochondria, where it physically interacts with and inactivates prosurvival Bcl-2 proteins. In the present study, we therefore investigated the role of mitochondrial translocation of p53 for the stress response of tumor cells. In various cell lines, DNA damage induced by either ionizing irradiation or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by ionizing radiation. Irradiated cells became senescent, although irradiation triggered a functional p53 response and induced expression of p21, Bax, and Puma. Interestingly, even the targeted expression of p53 to mitochondria was insufficient to launch apoptosis, whereas overexpression of wild-type p53 induced Bax activation and apoptotic alterations. Together, these results suggest that, in contrast to previous reports, mitochondrial translocation of p53 does not per se lead to cell death and that this might constitute a mechanism that contributes to the resistance of tumor cells to ionizing radiation-induced apoptosis.
Russian Journal of Genetics, 2009
Changes in the number of mutant copies of mitochondrial DNA (mtDNA) were studied in the brain and spleen tissues of mice after their X-irradiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA ( ND 3 gene and two D-loop regions) from irradiated and control mice were digested with the Cel I nuclease capable of specific mismatch cleavage. Heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA from irrradiated and control mice were digested by the Cel I nuclease to a greater degree than heteroduplexes of the PCR products of mtDNA of mice from the control group. This suggests the presence of mutations in mtDNA regions in irradiated mice. Digestion by the Cel I nuclease of heteroduplexes obtained via hybridization of the PCR products of mtDNA ( ND 3 gene and D-loop regions) on day 8 after irradiation is essentially more efficient than digestion of heteroduplexes obtained via hybridization of the PCR products of mtDNA isolated from mouse tissues on days 14 and 28 of the postradiation period. These results indicate a reduction in the number of mtDNA copies with mutations in tissues of irradiated mice by day 28 of the postradiation period. The reduction in the level of mutant mtDNA copies by this term is especially significant in the spleen. The total number of mtDNA copies in the mouse brain and spleen tissues estimated by real-time PCR, relative to the nuclear β -actin gene, is also decreased by 30-50% as compared to the control on days 8 to 28 after irradiation. The results of the study suggest that mutant mtDNA copies are eliminated from tissues of irradiated animals in the postradiation period. This elimination can be regarded either as a result of selective degradation of mitochondria carrying mutant DNA copies or as a result of cell death being continued in tissues of irradiated animals.
Replication of Murine Mitochondrial DNA Following Irradiation
The effect of radiation on the mitochondrial genome in vivois largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2005
Radiation damage incurred by nuclear DNA is well documented and interest is increasing in the properties of 'bystander' factor(s) and their ability to induce radiation-like damage in cells never exposed to radiation. 'Bystander' and direct low-LET radiation effects on the mitochondria, and more particularly the mitochondrial genome are less well understood. In this study HPV-G cells (a human keratinocyte cell line derived from human neonatal foreskin transfected with the HPV-16 virus) were exposed to either ␥-radiation doses as low as 5 mGy and up to 5 Gy from a 60 Co teletherapy unit, or to growth medium taken from similarly irradiated cells, i.e. irradiated cell conditioned medium (ICCM). Mutation and deletion analysis was performed on mitochondrial DNA (mtDNA) 4-96 h after exposure. Primers flanking the so-called mitochondrial 'common deletion' were employed to assess its possible induction. Single-strand conformation polymorphism (SSCP) analysis was conducted to identify induced point mutations. The relative mitochondrial number per cell was analysed by semi-quantitative PCR (sqPCR). Results indicate the induction of a relatively novel deletion in the mitochondrial genome as early as 12 h after direct exposure to doses as low as 0.5 Gy and 24 h after exposure to 0.5-Gy ICCM. SSCP analysis identified the induction of point mutations, in a non-consistent manner, in only the D-loop region of the mitochondrial genome and only in cells exposed to 5 Gy, and neither in cells exposed to lower doses of direct radiation nor in those exposed to ICCM. SqPCR also identified an increase in the number of mitochondria per cell after both exposure to low level ␥-radiation and ICCM, indicative of a possible mechanism to respond to mitochondrial stress by increasing the number of mitochondria per cell.
Molecular Biology, 2009
Nucleic acids circulating in blood plasma and other biological fluids are of interest as potential markers for the diagnosis of various pathologies and the monitoring of stresses. Mitochondrial DNA (mtDNA) is a more vulnerable target for many genotoxic agents than nuclear DNA, and mutations in the mitochondrial genome can serve as markers for many diseases. In the present study, extracellular mtDNA with mutations was assayed in the blood plasma of mice exposed to X radiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained by the hybridization of mtDNA PCR amplicons ( ND3 gene and D loop region) from the blood plasma of irradiated and control mice were cleaved with CEL endonuclease, a mismatch-specific enzyme. The total amount of mtDNA ( ND4 gene) copies vs. nuclear DNA ( GAPDH gene) was measured by real-time PCR. The content of mtDNA with mutations in murine blood plasma remained high within one month after irradiation but varied with time. The measurements were performed on days 1, 4, 8, 14, and 28 after irradiation, and the maximum level was detected on day 14. The elevated content of extracellular mutant mtDNA in blood plasma of X-irradiated mice is a sensitive candidate biomarker for the assessment of radiation injury and effects of other genotoxic agents.
2011
Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated β-galactosidase (SA-β-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and H2O2-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and H2O2-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-β-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.
International Journal of Radiation Biology, 2010
To further evaluate irregular mitochondrial function and mitochondrial genome damage induced by direct γ irradiation and bystander factors in human keratinocyte (HPV-G) epithelial cells and hamster ovarian fibroblast (CHO-K1) cells. This is as a follow up to our recent reports of γ-irradiation induced loss of mitochondrial function and mitochondrial DNA (mtDNA) damage. Materials and Methods: Mitochondrial function was evaluated post direct radiation and irradiated cell conditioned medium (ICCM) by determining: activity of the individual complexes of oxidative phosphorylation (OxPhos); mtDNA-encoded protein synthesis; mitochondrial genome frequency and mtDNA damage. Results: Mitochondria show a loss of OxPhos enzyme function as early as 4 hours post treatment with recovery observed 12-96 hours in some but not all complexes demonstrating a non-uniform sensitivity to γ radiation. We also identified irregular mtDNA-directed protein synthesis. Long range Polymerase Chain Reaction (PCR) analysis identified mitochondrial genome damage and real time PCR identified increases in mitochondrial genome frequency. Conclusions: The study reaffirms the sensitive nature of mitochondria to both low-level direct radiation exposure and radiation-induced bystander factor mediated damage. Furthermore, we report for the first time, the loss of function in the enzymes of OxPhos post exposure to bystander factors and identify altered mtDNA-directed protein synthesis post both direct radiation and bystander factors.
Cancer Research, 2007
The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiationinduced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-ρ 0 cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal ρ + cells.