Gene expression as a biomarker for human radiation exposure.

Accidental exposure to ionizing radiation can be unforeseen, rapid, and devastating. The detonation of a radiological device leading to such an exposure can be detrimental to the exposed population. The radiation-induced damage may manifest as acute effects that can be detected clinically or may be more subtle effects that can lead to long-term radiation-induced abnormalities. Accurate identification of the individuals exposed to radiation is challenging. The availability of a rapid and effective screening test that could be used as a biomarker of radiation exposure detection is mandatory. We tested the suitability of alterations in gene expression to serve as a biomarker of human radiation exposure. To develop a useful gene expression biomonitor, however, gene expression changes occurring in response to irradiation in vivo must be measured directly. Patients undergoing radiation therapy provide a suitable test population for this purpose. We examined the expression of CC3, MADH7, and SEC PRO in blood samples of these patients before and after radiotherapy to measure the in vivo response. The gene expression after ionizing radiation treatment varied among different patients, suggesting the complexity of the response. The expression of the SEC PRO gene was repressed in most of the patients. The MADH7 gene was found to be upregulated in most of the subjects and could serve as a molecular marker of radiation exposure.

Identification of radiation-induced microRNA transcriptome by next-generation massively parallel sequencing.

Gene regulation in cells exposed to ionizing radiation (IR) occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) play a significant role in post-transcriptional gene regulation in irradiated cells. miRNA are RNA molecules 18-24 nucleotides in length that are involved in negatively regulating the stability or translation of target messenger RNA. Previous studies from our laboratory have shown that the expression of various miRNA is altered in IR-treated cells. In the present study we monitored genome-wide expression changes of miRNA transcriptome by massively parallel sequencing of human cells irradiated with X-rays. The baseline expression of 402 miRNA indicated a wide range of modulation without exposure to IR. Differences in the expression of many miRNA were observed in a time-dependent fashion following radiation treatment. The Short Time-series Expression Miner (STEM) clustering tool was used to characterize 190 miRNA to six statistically significant temporal expression profiles. miR-19b and miR-93 were induced and miR-222, miR-92a, and miR-941 were repressed after radiation treatment. miR-142-3p, miR-142-5p, miR-107, miR-106b, miR-191, miR-21, miR-26a, miR-182, miR-16, miR-146a, miR-22 and miR-30e exhibited two peaks of induction: one at 8 h and the other at 24 h post-irradiation. miR-378, miR-let-7a, miR-let-7g, miR-let-7f, miR-103b, miR-486-3p, miR-423-5p, miR-4448, miR-3607-5p, miR-20b, miR-130b, miR-155, miR-181, miR-30d and miR-378c were induced only at the 8-h time-point. This catalogue of the inventory of miRNA that are modulated as a response to radiation exposure will be useful for explaining the mechanisms of gene regulation under conditions of stress.

Micro RNA responses to chronic or acute exposures to low dose ionizing radiation.

Human health risks of exposure to low dose ionizing radiation remain ambiguous and are the subject of intense debate. A wide variety of biological effects are induced after cellular exposure to ionizing radiation, but the underlying molecular mechanism(s) remain to be completely understood. We hypothesized that low dose γ-radiation-induced effects are controlled by the modulation of micro RNA (miRNA) that participate in the control of gene expression at the posttranscriptional level and are involved in many cellular processes. We monitored the expression of several miRNA in human cells exposed to acute or chronic low doses of 10 cGy or a moderate dose of 400 cGy of (137)Cs γ-rays. Dose, dose rate and time dependent differences in the relative expression of several miRNA were investigated. The expression patterns of many miRNA differed after exposure to either chronic or acute 10 cGy. The expression of miRNA let-7e, a negative regulator of RAS oncogene, and the c-MYC miRNA cluster were upregulated after 10 cGy chronic dose but were downregulated after 3 h of acute 10 cGy. The miR-21 was upregulated in chronic or acute low dose and moderate dose treated cells and its target genes hPDCD4, hPTEN, hSPRY2, and hTPM1 were found to be downregulated. These findings provide evidence that low dose and dose rate γ-irradiation dictate the modulation of miRNA, which can result in a differential cellular response than occurs at high doses. This information will contribute to understanding the risks to human health after exposure to low dose radiation.

Differential DNA methylation alterations in radiation-sensitive and -resistant cells.

An understanding of cellular processes that determine the response to ionizing radiation (IR) exposure is essential to improve radiotherapy and to assess risks to human health after accidental radiation exposure. Exposure to IR induces a multitude of biological effects. Recent studies have indicated the involvement of epigenetic events in regulating the responses of irradiated cells. DNA methylation, where the cytosine bases in CpG dimers are converted to 5-methyl cytosine, is an epigenetic event that has been shown to regulate a variety of biological processes. We investigated the DNA methylation changes in irradiated TK6 and WTK1 human cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an enzyme-linked immunosorbent assay-based assay showed hypomethylation in both type of cells. Using an arbitrarily primed polymerase chain reaction (AP-PCR) approach, we observed time-dependent dynamic changes in the regional genomic DNA methylation patterns in both cell lines. The AP-PCR DNA methylation profiles were different between TK6 and WTK1 cells, indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells after irradiation but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells after radiation treatment. TET1, involved in the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns, and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns change at different genomic regions and are dependent on time after irradiation and the genetic background of the cell.

Mitochondrial gene expression in directly irradiated and nonirradiated bystander cells.

Nontargeted cellular effects by ionizing radiation are well documented. The bystander effect is a nontargeted phenomenon wherein the irradiated cells communicate and induce changes in nonirradiated cells. The nature of the bystander signal and how it impacts unirradiated cells remain to be elucidated. Examination of molecular changes could lead to the identification of molecular pathways underlying the bystander effect. In this study, mitochondrial gene transcriptional changes in bystander cells were monitored to gain insight into the participation of mitochondria in this response. The modulation of mitochondrial gene expression in medium-exchanged bystander cells was determined in human lymphoblast TK6 cells by employing the real-time polymerase chain reaction technology. The examination of the relative expression of mitochondrial genes involved in various metabolic functions indicated that MT-ND1, MT-ND5, and MT-ND6 encoding NADH dehydrogenases were upregulated in directly irradiated cells but repressed in bystander cells. The differences in the expression levels were statistically significant among irradiated and bystander cells. The adenosine triphosphate (ATP) synthases MT-ATP6 and MT-ATP8 were upregulated in both irradiated and bystander cells. These results point to the involvement of mitochondrial gene modulation in directly irradiated and bystander cells and provide evidence that mitochondrial gene expression response is part of a complex stress response operating in radiation-treated cells.

Detection of genomic DNA methylation with denaturing high performance liquid chromatography.

DNA methylation contributes to the epigenetic control of gene expression. Variations in the methylation status can result in the silencing of genes. DNA methyltransferase converts cytosine to 5-methyl cytosine in CpG islands located in the promoter regions of genes. When CpG islands are hypermethylated, the gene is repressed/silenced, and similarly when it is hypomethylated, transcription can take place and the gene is expressed. The classical methods to detect DNA methylation require labor-intensive and time-consuming steps. As a result of large-scale expression profiling studies, high-throughput techniques are needed to screen for alterations in the methylation patterns. Denaturing high performance liquid chromatography (DHPLC) is a reliable, highly sensitive technique for mutation discovery. In the present study we examined the suitability of DHPLC technology to detect alterations in methylation pattern of the promoter regions of several genes. We report reliable and reproducible results in distinguishing methylated and unmethylated promoter regions of human PCDHGB6, c-MYC, MGMT1, CDKN2A/p16, and ATM genes. These DHPLC profiles were independently confirmed with bisulfite genomic sequencing. In conclusion, DHPLC technology serves as a rapid screening tool to monitor the genomic DNA methylation and could be used to increase the throughput efficiency of methylation analysis.

Transcriptional modulation of micro-RNA in human cells differing in radiation sensitivity.

PURPOSE: The molecular basis of gene regulation in cells exposed to ionising radiation is not fully understood. Gene regulation occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) plays a significant role at the post-transcriptional gene regulation. miRNA are a recently identified class of RNA molecules 18-24 nucleotides in length that have been shown to negatively regulate the stability or translation of target messenger RNA. We hypothesised that ionising radiation induced stress response is controlled in part by miRNA and that a difference in tumour protein 53 (p53) status corresponds with altered expression in miRNA responses to ionising radiation. MATERIALS AND METHODS: To test this hypothesis, we investigated the relative expression of several miRNA by quantitative real-time polymerase chain reaction (QPCR) in human cell lines TK6 and WTK1 that differ in p53 status and radiosensitivity after exposure to high and low doses of X-radiation. RESULTS: The suitability of several endogenous miRNA controls was tested for relative quantification by QPCR. The baseline expression of 21 miRNA targets in TK6 and WTK1 cells indicated a wide range of modulation between the two cell lines without exposure to ionising radiation. Differences in the relative expression of miRNA were observed among the two cell lines after radiation treatment. The expression patterns of many miRNA markedly differed within the same cell line after exposure to either 0.5 Gy or 2 Gy doses of X-rays. The expression of eight miRNA belonging to the lethal-7 (let-7) family, which are negative regulators of the rat sarcoma, RAS oncogene, was upregulated in irradiated TK6 cells but was downregulated in WTK1 cells. Alterations in the myelocytomatosis oncogene, c-MYC induced cluster of miRNA were also observed. The micro RNA, miR-15a and miR-16 were upregulated in 0.5 Gy-irradiated TK6 cells but were downregulated after a 2 Gy dose of X-rays. In contrast miR-15 and miR-16 were repressed in 0.5 Gy-exposed WTK1. The miR-21 was upregulated in 0.5 Gy-treated TK6 cells and its target genes programmed cell death factor 4 (hPDCD4) phosphatase and tensin homolog (hPTEN), and sprouty homolog 2 (hSPRY2) were found to be downregulated in these cells. The miR-21 was downregulated in 2 Gy-irradiated TK6 cells, and all three of its target genes were upregulated in 2 Gy-exposed TK6 cells. CONCLUSION: Taken together, these results establish the involvement of miRNA in radiation response and may potentially help explain the mechanisms of gene regulation in the cellular response to ionising radiation exposure.

Radiation-induced micro-RNA modulation in glioblastoma cells differing in DNA-repair pathways.

Human glioblastomas often develop resistance to radiation therapy. The molecular details of this phenomenon are not completely understood. Recent studies have suggested that deficiency in DNA repair pathways may alter the resistance to ionizing radiation in gliobastomas. The human glioma cell line M059J is deficient in DNA-dependent protein kinase (DNA-PK), whereas cell line M059K, isolated from the same malignant tumor, has normal DNA-PK activity. DNA-PK plays a central role in the repair of ionizing-radiation-induced double-strand break repair, and its deficiency has been correlated with ionizing radiation sensitivity in these glioblastoma cells. We argued that other cellular pathways could also play a role in the resistance to radiation therapy in gliomas. We hypothesized that micro-RNAs (miRNAs) are differentially modulated in M059J and M059K cells exposed to ionizing radiation and that the miRNA modulation contributes to the resistance to ionizing radiation. miRNAs are small nonprotein coding single-stranded RNA molecules, which are crucial posttranscriptional regulators of gene expression. Numerous studies have documented the participation of miRNAs in a wide range of biological processes. The contribution of miRNAs in mediating resistance of glioblastoma cell to ionizing radiation treatment has not been elucidated. To test this hypothesis, we examined the expression patterns of a number of miRNAs involved in carcinogenesis in irradiated M059J and M059K cells. The relative expression level as determined by real-time quantitative PCR for miRNAs belonging to the let-7 family indicated an upregulation in irradiated M059K cells. On the contrary, the analysis of irradiated M059J cells for the modulation of let-7 family of miRNAs revealed an overall downregulation. The miR-17-3p, miR-17-5p, miR-19a, miR-19b, miR-142-3p, and miR-142-5p were upregulated in both M059K and M059J cells. The miR-15a, miR-16, miR-143, miR-155, and miR-21 were upregulated in M059K, and the modulation of these miRNAs fluctuated in M059J cells in a time-dependent manner. These results indicate the involvement of miRNAs in the differential response of glioblastoma cells to ionizing radiation treatment.