Search Results (11)

The ferredoxin reductase (FDXR) gene is expressed as seven isoforms: 1–6 by alternative splicing and 7 by an alternative promoter according to the Entrez Gene Database. Previous studies showed that FDXR, primarily the mitochondrial isoform 1, plays a role in biosynthesis of sterols, heme, and iron–sulfur clusters. However, the biological functions of FDXR isoforms 3–7 have not been characterized. Here, we first examined the expression profile of various FDXR isoforms. We found that isoform 1 is the most abundant one, accounting for ~70% of total FDXR, whereas isoforms 4 and 7 account for ~10% and ~7%, respectively. We found that isoforms 1 and 4 are mainly localized in the mitochondria, whereas isoform 7, which lacks a mitochondria localization signal (MLS), is expressed in the cytosol. We also found that like the promoter 1 for isoforms 1-6, the P2 promoter for isoform 7 can be induced by DNA damage in a p53-dependent manner. To determine isoform-specific activity, we generated multiple MCF7 cell lines in which one or more FDXR isoforms are knocked out. While total FDXR-KO MCF7 cells are non-viable, cells deficient in isoforms 1–6, isoform 4, or isoform 7 remain viable but are defective in cell proliferation, DNA damage response, and repair. These data suggest that each FDXR isoform contributes to cell survival and that isoform 7 has extra-mitochondrial activity that may be sufficient for cell survival. Full article

Computed tomography (CT) is a major source of low-dose ionizing radiation exposure in medical imaging. Risk assessment at this dose level is difficult and relies on the hypothetical linear no-threshold model. To address the response to such low doses in patients undergoing CT scans, we examined radiation-induced alterations at the transcriptomic and DNA damage levels in peripheral blood cells. Peripheral whole blood of 60 patients was collected before and after CT. Post-CT samples were obtained 4–6 h after scan (n = 28, in vivo incubation) or alternatively immediately after the CT scan, followed by ex vivo incubation (n = 32). The gene expression of known radiation-responsive genes (n = 9) was quantified using qRT-PCR. DNA double-strand breaks (DSB) were assessed in 12 patients through microscopic γ-H2AX + 53BP1 DSB focus staining. The mean dose–length product (DLP) across all scans was 561.9 ± 384.6 mGy·cm. Significant differences in the median differential gene expression (DGE) were detected between in vivo and ex vivo incubation conditions, implicating that ex vivo incubation masked the true effect in low-dose settings. The median DGE of in vivo-incubated samples showed a significant upregulation of EDA2R, MIR34AHG, PHLDA3, DDB2, FDXR, and AEN (p ranging from <0.001 to 0.041). In vivo, we observed a linear dose-dependent upregulation for several genes and an explained variance of 0.66 and 0.56 for AEN and FDXR, respectively. DSB focus analysis revealed a slight, non-significant increase in the average DSB damage post-exposure, at a mean DLP of 321.0 mGy·cm. Our findings demonstrate that transcriptional biomarkers are sensitive indicators of low-dose radiation exposure in medical imaging and could prove themselves as clinically applicable biodosimetry tools. Furthermore, the results underscore the need for dose optimization. Full article

The increasing use of computed tomography (CT) has led to a rise in cumulative radiation dose due to medical imaging, raising concerns about potential long-term adverse effects. Large-scale epidemiological studies indicate a higher tumor incidence associated with CT examinations, but the underlying biological mechanisms remain largely unexplained. To gain further insights into the cellular response triggered by routine CT diagnostics, we investigated CT-induced changes of gene expression in peripheral blood cells using whole transcriptome sequencing. RNA was isolated from peripheral blood cells of 40 male patients with asymptomatic microhematuria, sampled before and after multi-phase abdominal CT (CTDIvol: 3.75–26.95 mGy, median: 6.55 mGy). On average, 22.11 million sequence reads (SD 5.71) per sample were generated to identify differentially expressed genes 6 h post-exposure by means of DESeq2. To assess the dose dependency of CT-induced effects, we additionally divided samples into three categories: low exposure (≤6.55 mGy, n = 20), medium exposure (>6.55 mGy and <12 mGy, n = 16), and high exposure (≥12 mGy, n = 4), and repeated gene expression analysis for each subset and their corresponding prae-exposure sample. CT exposure caused consistent and dose-dependent upregulation of six genes (EDA2R, AEN, FDXR, DDB2, PHLDA3, and MIR34AHG; padj < 0.1). These genes share several functional commonalities, including regulation by TP53 and involvement in the DNA damage response. The biological pathways highlighted by Gene Set Enrichment Analysis (GSEA) suggest a dose-dependent increase of cellular damage and metabolic particularities in the low-exposure subset, which may be related to a potential adaptive cellular response to low-dose irradiation. Irrespective of applied dose, AEN emerged as the most robust biomarker for CT exposure among all genes. Routine abdominal CT scans cause dose-dependent gene deregulation in association with DNA damage in peripheral blood cells after in vivo exposure. Regarding risk assessment of CT, our results support the commonly applied “As Low–As –Reasonably Achievable (ALARA)” principle. Evidence of additional gene expression changes associated with metabolic processes indicates a rather complex molecular response beyond DNA damage after CT exposure, and emphasizes the need for further targeted investigations. Full article

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenemia of ovarian thecal cell origin, resulting in anovulation/oligo-ovulation and infertility. Our previous studies established that ovarian theca cells isolated and propagated from ovaries of normal ovulatory women and women with PCOS have distinctive molecular and cellular signatures that underlie the increased androgen biosynthesis in PCOS. To evaluate differences between gene expression in single-cells from passaged cultures of theca cells from ovaries of normal ovulatory women and women with PCOS, we performed single-cell RNA sequencing (scRNA-seq). Results from these studies revealed differentially expressed pathways and genes involved in the acquisition of cholesterol, the precursor of steroid hormones, and steroidogenesis. Bulk RNA-seq and microarray studies confirmed the theca cell differential gene expression profiles. The expression profiles appear to be directed largely by increased levels or activity of the transcription factors SREBF1, which regulates genes involved in cholesterol acquisition (LDLR, LIPA, NPC1, CYP11A1, FDX1, and FDXR), and GATA6, which regulates expression of genes encoding steroidogenic enzymes (CYP17A1) in concert with other differentially expressed transcription factors (SP1, NR5A2). This study provides insights into the molecular mechanisms underlying the hyperandrogenemia associated with PCOS and highlights potential targets for molecular diagnosis and therapeutic intervention. Full article

Variants in FDXR reportedly cause autosomal recessive auditory neuropathy and optic atrophy, expanding to retinal dystrophy. This study aimed to further clarify associated phenotypes. FDXR variants were selected from our in-house whole-exome sequencing dataset of 6397 families with different eye conditions. The clinical data of the identified patients were summarized. Biallelic pathogenic or likely pathogenic FDXR variants were identified in 11 unrelated patients, including 14 missense variants of which 10 were novel. Fundus observation showed complete optic disc pallor, silver wiring or severe attenuation of retinal vessels, and varying degrees of generalized retinal degeneration. Before the detection of FDXR variants, four patients were clinically diagnosed as congenital amaurosis due to the presence of nystagmus a few months after birth, while seven were diagnosed as early-onset severe retinal dystrophy due to the presence of nyctalopia and/or poor vision in early childhood. Biallelic FDXR variants are a frequent cause of congenital or early-onset severe retinal dystrophy, especially for patients with severe optic atrophy and retinal dystrophy in early childhood. Full article

To assess the prognostic value of “liquid biopsies” for the benefit of salvage RT in oligometastatic prostate cancer relapse, we enrolled 44 patients in the study between the years 2016 and 2020. All the patients were diagnosed as having an oligometastatic prostate cancer relapse on prostate-specific membrane antigen (PSMA)-targeted PET-CT and underwent irradiation at the Department of Radiotherapy at the Hannover Medical School. Tumor cells and total RNA, enriched from the liquid biopsies of patients, were processed for the subsequent quantification analysis of relative transcript levels in real-time PCR. In total, 54 gene transcripts known or suggested to be associated with prostate cancer or treatment outcome were prioritized for analysis. We found significant correlations between the relative transcript levels of several investigated genes and the Gleason score, PSA (prostate-specific antigen) value, or UICC stage (tumor node metastasis -TNM classification of malignant tumors from Union for International Cancer Control). Furthermore, a significant association of MTCO2, FOXM1, SREBF1, HOXB7, FDXR, and MTRNR transcript profiles was found with a temporary and/or long-term benefit from RT. Further studies on larger patients cohorts are necessary to prove our preliminary findings for establishing liquid biopsy tests as a predictive examination method prior to salvage RT. Full article

Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest ovarian cancer stem cells (OCSCs) contribute to chemotherapy resistance and tumor relapse. Recent studies demonstrated estrogen receptor beta (ERβ) exerts tumor suppressor functions in OCa. However, the status of ERβ expression in OCSCs and the therapeutic utility of the ERβ agonist LY500307 for targeting OCSCs remain unknown. OCSCs were enriched from ES2, OV90, SKOV3, OVSAHO, and A2780 cells using ALDEFLUOR kit. RT-qPCR results showed ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs and that ERβ agonist LY500307 significantly reduced the viability of OCSCs. Treatment of OCSCs with LY500307 significantly reduced sphere formation, self-renewal, and invasion, while also promoting apoptosis and G2/M cell cycle arrest. Mechanistic studies using RNA-seq analysis demonstrated that LY500307 treatment resulted in modulation of pathways related to cell cycle and apoptosis. Western blot and RT-qPCR assays demonstrated the upregulation of apoptosis and cell cycle arrest genes such as FDXR, p21/CDKN1A, cleaved PARP, and caspase 3, and the downregulation of stemness markers SOX2, Oct4, and Nanog. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopic OCa murine xenograft models. Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and shows significant promise as a novel therapeutic agent in treating OCa. Full article

External beam radiation therapy leads to cellular activation of the DNA damage response (DDR). DNA double-strand breaks (DSBs) activate the ATM/CHEK2/p53 pathway, inducing the transcription of stress genes. The dynamic nature of this transcriptional response has not been directly observed in vivo in humans. In this study we monitored the messenger RNA transcript abundances of nine DNA damage-responsive genes (CDKN1A, GADD45, CCNG1, FDXR, DDB2, MDM2, PHPT1, SESN1, and PUMA), eight of them regulated by p53 in circulating blood leukocytes at different time points (2, 6–8, 16–18, and 24 h) in cancer patients (lung, neck, brain, and pelvis) undergoing radiotherapy. We discovered that, although the calculated mean physical dose to the blood was very low (0.038–0.169 Gy), an upregulation of Ferredoxin reductase (FDXR) gene transcription was detectable 2 h after exposure and was dose dependent from the lowest irradiated percentage of the body (3.5% whole brain) to the highest, (up to 19.4%, pelvic zone) reaching a peak at 6–8 h. The radiation response of the other genes was not strong enough after such low doses to provide meaningful information. Following multiple fractions, the expression level increased further and was still significantly up-regulated by the end of the treatment. Moreover, we compared FDXR transcriptional responses to ionizing radiation (IR) in vivo with healthy donors’ blood cells exposed ex vivo and found a good correlation in the kinetics of expression from the 8-hours time-point onward, suggesting that a molecular transcriptional regulation mechanism yet to be identified is involved. To conclude, we provided the first in vivo human report of IR-induced gene transcription temporal response of a panel of p53-dependant genes. FDXR was demonstrated to be the most responsive gene, able to reliably inform on the low doses following partial body irradiation of the patients, and providing an expression pattern corresponding to the % of body exposed. An extended study would provide individual biological dosimetry information and may reveal inter-individual variability to predict radiotherapy-associated adverse health outcomes. Full article

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors. Full article

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers. Full article

Accumulating evidence suggests a synergistic effect in cells simultaneously exposed to different types of clustered and dispersed DNA damage. We aimed to analyse the effect of mixed beams of alpha particles and X-rays (1:1 dose of each) on DNA damage response genes in human peripheral blood lymphocytes isolated from four donors. Two donors were compared upon inhibition of ATM or DNA-PK and at different sampling times. qPCR was used to measure mRNA levels of FDXR, GADD45A, BBC3, MDM2, CDKN1A, and XPC 24 h following exposure. Generally, alpha particles and mixed beams were stronger inducers of gene expression compared to X-rays, displaying saturated versus linear dose–response curves, respectively. Three out of four donors responded synergistically to mixed beams. When two donors were sampled again one year later, the former additive effect in one donor was now synergistic and no significant difference in intrinsic radiosensitivity was displayed, as determined by gamma-radiation-induced micronuclei. ATM, but not DNA-PK inhibition, reduced the radiation-induced gene expression, but differently for alpha radiation between the two donors. In conclusion, synergy was present for all donors, but the results suggest individual variability in the response to mixed beams, most likely due to lifestyle changes. Full article