HEMOPOIETIC RESPONSE TO LOW DOSE-RATES OF IONIZING RADIATION SHOWS STEM CELL TOLERANCE AND ADAPTATION

Chronic exposure of mammals to low dose-rates of ionizing radiation affects proliferating cell systems as a function of both dose-rate and the total dose accumulated. The lower the dose-rate the higher needs to be the total dose for a deterministic effect, i.e., tissue reaction to appear. Stem cells provide for proliferating, maturing and functional cells. Stem cells usually are particularly radiosensitive and damage to them may propagate to cause failure of functional cells. The paper revisits 1) medical histories with emphasis on the hemopoietic system of the victims of ten accidental chronic radiation exposures, 2) published hematological findings of long-term chronically gamma-irradiated rodents, and 3) such findings in dogs chronically exposed in large life-span studies. The data are consistent with the hypothesis that hemopoietic stem and early progenitor cells have the capacity to tolerate and adapt to being repetitively hit by energy deposition events. The data are compatible with the “injured stem cell hypothesis”, stating that radiation–injured stem cells, depending on dose-rate, may continue to deliver clones of functional cells that maintain homeostasis of hemopoiesis throughout life. Further studies perhaps on separated hemopoietic stem cells may unravel the molecular-biology mechanisms causing radiation tolerance and adaptation

Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

<p>Abstract</p> <p>Background</p> <p>We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.</p> <p>Methods</p> <p>Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.</p> <p>Results</p> <p>Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).</p> <p>Conclusion</p> <p>Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.</p

Responses of Cell Renewal Systems to Long-term Low-Level Radiation Exposure: A Feasibility Study Applying Advanced Molecular Biology Techniques on Available Histological and Cytological Material of Exposed Animals and Men

First results of this feasibility study showed that evaluation of the stored material of the chronically irradiated dogs with modern molecular biological techniques proved to be successful and extremely promising. Therefore an in deep analysis of at least part of the huge amount of remaining material is of outmost interest. The methods applied in this feasibility study were pathological evaluation with different staining methods, protein analysis by means of immunohistochemistry, strand break analysis with the TdT-assay, DNA- and RNA-analysis as well as genomic examination by gene array. Overall more than 50% of the investigated material could be used. In particular the results of an increased stimulation of the immune system within the dogs of the 3mSv group as both compared to the control and higher dose groups gives implications for the in depth study of the cellular events occurring in context with low dose radiation. Based on the findings of this study a further evaluation and statistically analysis of more material can help to identify promising biomarkers for low dose radiation. A systematic evaluation of a correlation of dose rates and strand breaks within the dog tissue might moreover help to explain mechanisms of tolerance to IR. One central problem is that most sequences for dog specific primers are not known yet. The discovery of the dog genome is still under progress. In this study the isolation of RNA within the dog tissue was successful. But up to now there are no gene arrays or gene chips commercially available, tested and adapted for canine tissue. The uncritical use of untested genomic test systems for canine tissue seems to be ineffective at the moment, time consuming and ineffective. Next steps in the investigation of genomic changes after IR within the stored dog tissue should be limited to quantitative RT-PCR of tested primer sequences for the dog. A collaboration with institutions working in the field of the discovery of the dog genome could have synergistic effects

Small RNAs in the peripheral blood discriminate metastasized from non-metastasized seminoma

Background: We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood. Methods: Total RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours (lymphogen, n = 5, clinical stage IIb/c; occult, n = 5, clinical stage I) and non-metastasized patients (n = 5, clinical stage I). Small RNA next generation sequencing (SOLID, Life Technologies) was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant >= 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. Results: On average 1.3x10(7), 1.2x10(7) and 1.2x10(7) small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7x10(4)) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a >= two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype. Conclusions: Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood

Acute radiation syndrome caused by accidental radiation exposure - therapeutic principles

Fortunately radiation accidents are infrequent occurrences, but since they have the potential of large scale events like the nuclear accidents of Chernobyl and Fukushima, preparatory planning of the medical management of radiation accident victims is very important. Radiation accidents can result in different types of radiation exposure for which the diagnostic and therapeutic measures, as well as the outcomes, differ. The clinical course of acute radiation syndrome depends on the absorbed radiation dose and its distribution. Multi-organ-involvement and multi-organ-failure need be taken into account. The most vulnerable organ system to radiation exposure is the hematopoietic system. In addition to hematopoietic syndrome, radiation induced damage to the skin plays an important role in diagnostics and the treatment of radiation accident victims. The most important therapeutic principles with special reference to hematopoietic syndrome and cutaneous radiation syndrome are reviewed

Autologous Adipocyte Derived Stem Cells Favour Healing in a Minipig Model of Cutaneous Radiation Syndrome

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a 60Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL−1) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50×106 ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/− 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS

Persistent DNA Damage after High Dose In Vivo Gamma Exposure of Minipig Skin

Exposure to high doses of ionizing radiation (IR) can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin.IR-induced DNA damage, repair and cellular survival were studied in 15 cm(2) of minipig skin exposed in vivo to ~50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF) formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of <1% of keratinocytes at 28-70 days. The latter contained large RIFs that included ATM-p, indicating the accumulation of complex DNA damage. At 96 days most of the cells with RIFs had disappeared. The frequency of active-caspase-3-positive apoptotic cells was 17-fold increased 3 days after IR and remained >3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+) were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days.Acute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in <1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios