Identification of Genetic Predispositions Related to Ionizing Radiation in Primary Human Skin Fibroblasts From Survivors of Childhood and Second Primary Cancer as Well as Cancer-Free Controls: Protocol for the Nested Case-Control Study KiKme.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32395.

Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (IK1 ). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite8 ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.