Exposure to ionizing radiation induces complex stress responses in cells, which can lead to adverse health effects such as cancer. Although a variety of studies investigated gene expression and ...affected pathways in human fibroblasts after exposure to ionizing radiation, the understanding of underlying mechanisms and biological effects is still incomplete due to different experimental settings and small sample sizes. Therefore, this study aims to identify the time point with the highest number of differentially expressed genes and corresponding pathways in primary human fibroblasts after irradiation at two preselected time points.
Fibroblasts from skin biopsies of 15 cell donors were exposed to a high (2Gy) and a low (0.05Gy) dose of X-rays. RNA was extracted and sequenced 2 h and 4 h after exposure. Differentially expressed genes with an adjusted p-value < 0.05 were flagged and used for pathway analyses including prediction of upstream and downstream effects. Principal component analyses were used to examine the effect of two different sequencing runs on quality metrics and variation in expression and alignment and for explorative analysis of the radiation dose and time point of analysis.
More genes were differentially expressed 4 h after exposure to low and high doses of radiation than after 2 h. In experiments with high dose irradiation and RNA sequencing after 4 h, inactivation of the FAT10 cancer signaling pathway and activation of gluconeogenesis I, glycolysis I, and prostanoid biosynthesis was observed taking p-value (< 0.05) and (in) activating z-score (≥2.00 or ≤ - 2.00) into account. Two hours after high dose irradiation, inactivation of small cell lung cancer signaling was observed. For low dose irradiation experiments, we did not detect any significant (p < 0.05 and z-score ≥ 2.00 or ≤ - 2.00) activated or inactivated pathways for both time points.
Compared to 2 h after irradiation, a higher number of differentially expressed genes were found 4 h after exposure to low and high dose ionizing radiation. Differences in gene expression were related to signal transduction pathways of the DNA damage response after 2 h and to metabolic pathways, that might implicate cellular senescence, after 4 h. The time point 4 h will be used to conduct further irradiation experiments in a larger sample.
Therapy for a first primary neoplasm (FPN) in childhood with high doses of ionizing radiation is an established risk factor for second primary neoplasms (SPN). An association between exposure to low ...doses and childhood cancer is also suggested; however, results are inconsistent. As only subgroups of children with FPNs develop SPNs, an interaction between radiation, genetic, and other risk factors is presumed to influence cancer development.
Therefore, the population-based, nested case-control study KiKme aims to identify differences in genetic predisposition and radiation response between childhood cancer survivors with and without SPNs as well as cancer-free controls.
We conducted a population-based, nested case-control study KiKme. Besides questionnaire information, skin biopsies and saliva samples are available. By measuring individual reactions to different exposures to radiation (eg, 0.05 and 2 Gray) in normal somatic cells of the same person, our design enables us to create several exposure scenarios for the same person simultaneously and measure several different molecular markers (eg, DNA, messenger RNA, long noncoding RNA, copy number variation).
Since 2013, 101 of 247 invited SPN patients, 340 of 1729 invited FPN patients, and 150 of 246 invited cancer-free controls were recruited and matched by age and sex. Childhood cancer patients were additionally matched by tumor morphology, year of diagnosis, and age at diagnosis. Participants reported on lifestyle, socioeconomical, and anthropometric factors, as well as on medical radiation history, health, and family history of diseases (n=556). Primary human fibroblasts from skin biopsies of the participants were cultivated (n=499) and cryopreserved (n=3886). DNA was extracted from fibroblasts (n=488) and saliva (n=510).
This molecular-epidemiological study is the first to combine observational epidemiological research with standardized experimental components in primary human skin fibroblasts to identify genetic predispositions related to ionizing radiation in childhood and SPNs. In the future, fibroblasts of the participants will be used for standardized irradiation experiments, which will inform analysis of the case-control study and vice versa. Differences between participants will be identified using several molecular markers. With its innovative combination of experimental and observational components, this new study will provide valuable data to forward research on radiation-related risk factors in childhood cancer and SPNs.
DERR1-10.2196/32395.
Abstract
Treatment of first primary neoplasms (PN) in childhood with radiotherapy or chemotherapy is an established risk factor for second primary neoplasms (SN). In addition, there is growing ...evidence for this association from observational studies on ionizing radiation and cancer risk, in particular after radiation exposure in childhood. As only a subgroup of the treated children suffers from SN, other risk modifying factors (e.g. genetics) must be involved. We are conducting a case-control study with 600 anticipated participants to evaluate gene-radiation interactions and risk of SN (leukemia, thyroid or skin cancer) as well as PN (leukemia, lymphoma or CNS) with a new epidemiological design, in which we combine observational with experimental elements by analyzing gene expression in irradiated cultured human fibroblasts from skin biopsies.
In a first step, we examine the participation proportions of survivors of childhood cancer with and without a SN and cancer free control patients (CO) from the department of accident surgery and orthopaedics. In addition to a skin biopsy of 3 mm and a saliva sample, we collect detailed questionnaire information on lifetime exposure to medical radiation and chemotherapy, socio-demographic factors, smoking, drinking, physical activity, medical history and family history of cancer and other diseases. Cases and controls will be matched by sex and age (1:1), and additionally among the former childhood cancer patients by type of the PN and year of first diagnosis (1 SN:3 PN). In explorative pilot experiments, we estimate gene expression differences by RNA-Seq in fibroblasts after low (0.05 Gy) and high (2 Gy) radiation doses at different time points (0.25 h, 2 h, 24 h).
In the first recruitment drives of the ongoing study, we recruited 77 patients with SN and 95 matched patients with only one PN from 1975 eligible former childhood cancer patients at the German Childhood Cancer Registry, as well as 22 CO patients. Until November 2016, 33% of the contacted 231 SN patients, 20% of the 486 contacted PN patients and 69% of the 32 contacted CO patients participated in our study. Two hours after low and high in vitro radiation doses, the largest number of genes were differentially expressed, some of them only after high doses, some only after low doses and some after both.
To our knowledge, the KIKME study is the first epidemiological project analyzing differential gene expression in primary fibroblasts before and after radiation with high and low doses to evaluate the potential genetic basis for emergence of a SN and a PN. However, the biological importance of the suggested differential gene expression after high and low doses of radiation has to be confirmed with the full study population. In addition, the gene expression must be analyzed in detail by group (SN, PN, CO) and will be combined with results from whole genome sequencing in order to obtain a comprehensive view of the role of radiation in the carcinogenesis of childhood cancer.
Citation Format: Manuela Marron, Sebastian Zahnreich, Olesja Sinizyn, Heinz Schmidberger, Moritz Hess, Patricia Sadre Dadras, Iris Altebockwinkel, Thomas Hankeln, Steffen Rapp, Anne Ebersberger, Christian Grad, Eva Holzhäuser, Lukas Eckhard, Dirk Proschek, Maria Blettner, Peter Kaatsch, Claudia Spix, Danuta Galetzka, Harald Binder. Cancer in childhood and molecular epidemiology - The KIKME case-control study abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2017-4261
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