"Lethal Mutations" a Misnomer or the Start of a Scientific Revolution?

The aim of this paper is to review the history surrounding the discovery of lethal mutations, later described as delayed reproductive death. Lethal mutations were suggested very early on, to be due to a generalised instability in a cell population and are considered now to be one of the first demonstrations of "radiation-induced genomic instability" which led later to the establishment of the field of "non-targeted effects." The phenomenon was first described by Seymour et al. in 1986 and was confirmed by Trott's group in Europe and by Little and colleagues in the United States before being extended by Mendonca et al. in 1989, who showed conclusively that the distinguishing feature of lethal mutation occurrence was that it happened suddenly after about 9-10 population doublings in progeny which had survived the original dose of ionizing radiation. However, many authors then suggested that in fact, lethal mutations were implicit in the original experiments by Puck and Marcus in 1956 and were described in the extensive work by Sinclair in 1964, who followed clonal progeny for up to a year after irradiation and described "small colony formation" as a persistent consequence of ionizing radiation exposure. In this paper, we examine the history from 1956 to the present using the period from 1986-1989 as an anchor point to reach into the past and to go forward through the evolution of the field of low dose radiobiology where non-targeted effects predominate.

The REPAIR Project, a Deep-Underground Radiobiology Experiment Investigating the Biological Effects of Natural Background Radiation: The First 6 Years.

In 2017, a special edition of Radiation Research was published [Oct; Vol. 188 4.2 (https://bioone.org/journals/radiation-research/volume-188/issue-4.2)] which focused on a recently established radiobiology project within SNOLAB, a unique deep-underground research facility. This special edition included original articles, reviews and commentaries relevant to the research goals of this new project which was titled Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR). These research goals were founded in understanding the biological effects of terrestrial and cosmic natural background radiation (NBR). Since 2017, REPAIR has evolved into a sub-NBR radiobiology research program which investigates these effects using multiple model systems and various biological endpoints. This paper summarizes the evolution of the REPAIR project over the first 6-years including its experimental scope and capabilities as well as research accomplishments.

Overexpression of FRA1 (FOSL1) Leads to Global Transcriptional Perturbations, Reduced Cellular Adhesion and Altered Cell Cycle Progression.

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2-3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression.

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have exhibited great promise in the treatment of tumors with homologous recombination (HR) deficiency, however, PARPi resistance, which ultimately recovers DNA repair and cell progress, has become an enormous clinical challenge. Recently, KP372-1 was identified as a novel potential anticancer agent that targeted the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce extensive reactive oxygen species (ROS) generation that amplified DNA damage, leading to cancer cell death. To overcome PARPi resistance and expand its therapeutic utility, we investigated whether a combination therapy of a sublethal dose of KP372-1 with a nontoxic dose of PARPi rucaparib would synergize and enhance lethality in NQO1 over-expressing cancers. We reported that the combination treatment of KP372-1 and rucaparib induced a transient and dramatic AKT hyperactivation that inhibited DNA repair by regulating FOXO3a/GADD45α pathway, which enhanced PARPi lethality and overcame PARPi resistance. We further found that PARP inhibition blocked KP372-1-induced PARP1 hyperactivation to reverse NAD+/ATP loss that promoted Ca2+-dependent autophagy and apoptosis. Moreover, pretreatment of cells with BAPTA-AM, a cytosolic Ca2+ chelator, dramatically rescued KP372-1- or combination treatment-induced lethality and significantly suppressed PAR formation and γH2AX activation. Finally, we demonstrated that this combination therapy enhanced accumulation of both agents in mouse tumor tissues and synergistically suppressed tumor growth in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1+ solid tumors that may broaden the clinical utility of PARPi.

News FLASH-RT: To Treat GBM and Spare Cognition, Fraction Size and Total Dose Matter.

Data indicate that ultrahigh dose rate (>106 Gy/second) FLASH radiotherapy (FLASH-RT) delivery of radiation reduces normal tissue damage without compromising tumor response. Orthotopic glioblastoma mouse studies now demonstrate that radiation fraction size, total dose, and number of fractions are critical parameters for FLASH-RT cognitive sparing without compromising tumor response.See related article by Montay-Gruel et al., p. 775.

PCNA inhibition enhances the cytotoxicity of ß-lapachone in NQO1-Positive cancer cells by augmentation of oxidative stress-induced DNA damage.

ß-Lapachone is a classic quinone-containing antitumor NQO1-bioactivatable drug that directly kills NQO1-overexpressing cancer cells. However, the clinical applications of ß-lapachone are primarily limited by its high toxicity and modest lethality. To overcome this side effect and expand the therapeutic utility of ß-lapachone, we demonstrate the effects of a novel combination therapy including ß-lapachone and the proliferating cell nuclear antigen (PCNA) inhibitor T2 amino alcohol (T2AA) on various NQO1+ cancer cells. PCNA has DNA clamp processivity activity mediated by encircling double-stranded DNA to recruit proteins involved in DNA replication and DNA repair. In this study, we found that compared to monotherapy, a nontoxic dose of the T2AA synergized with a sublethal dose of ß-lapachone in an NQO1-dependent manner and that combination therapy prevented DNA repair, increased double-strand break (DSB) formation and promoted programmed necrosis and G1 phase cell cycle arrest. We further determined that combination therapy enhanced antitumor efficacy and prolonged survival in Lewis lung carcinoma (LLC) xenografts model. Our findings show novel evidence for a new therapeutic approach that combines of ß-lapachone treatment with PCNA inhibition that is highly effective in treating NQO1+ solid tumor cells.

Therapy-Induced Senescence: Opportunities to Improve Anticancer Therapy.

Cellular senescence is an essential tumor suppressive mechanism that prevents the propagation of oncogenically activated, genetically unstable, and/or damaged cells. Induction of tumor cell senescence is also one of the underlying mechanisms by which cancer therapies exert antitumor activity. However, an increasing body of evidence from preclinical studies demonstrates that radiation and chemotherapy cause accumulation of senescent cells (SnCs) both in tumor and normal tissue. SnCs in tumors can, paradoxically, promote tumor relapse, metastasis, and resistance to therapy, in part, through expression of the senescence-associated secretory phenotype. In addition, SnCs in normal tissue can contribute to certain radiation- and chemotherapy-induced side effects. Because of its multiple roles, cellular senescence could serve as an important target in the fight against cancer. This commentary provides a summary of the discussion at the National Cancer Institute Workshop on Radiation, Senescence, and Cancer (August 10-11, 2020, National Cancer Institute, Bethesda, MD) regarding the current status of senescence research, heterogeneity of therapy-induced senescence, current status of senotherapeutics and molecular biomarkers, a concept of "one-two punch" cancer therapy (consisting of therapeutics to induce tumor cell senescence followed by selective clearance of SnCs), and its integration with personalized adaptive tumor therapy. It also identifies key knowledge gaps and outlines future directions in this emerging field to improve treatment outcomes for cancer patients.

Physics and biology of ultrahigh dose-rate (FLASH) radiotherapy: a topical review.

Ultrahigh dose-rate radiotherapy (RT), or 'FLASH' therapy, has gained significant momentum following various in vivo studies published since 2014 which have demonstrated a reduction in normal tissue toxicity and similar tumor control for FLASH-RT when compared with conventional dose-rate RT. Subsequent studies have sought to investigate the potential for FLASH normal tissue protection and the literature has been since been inundated with publications on FLASH therapies. Today, FLASH-RT is considered by some as having the potential to 'revolutionize radiotherapy'. FLASH-RT is considered by some as having the potential to 'revolutionize radiotherapy'. The goal of this review article is to present the current state of this intriguing RT technique and to review existing publications on FLASH-RT in terms of its physical and biological aspects. In the physics section, the current landscape of ultrahigh dose-rate radiation delivery and dosimetry is presented. Specifically, electron, photon and proton radiation sources capable of delivering ultrahigh dose-rates along with their beam delivery parameters are thoroughly discussed. Additionally, the benefits and drawbacks of radiation detectors suitable for dosimetry in FLASH-RT are presented. The biology section comprises a summary of pioneering in vitro ultrahigh dose-rate studies performed in the 1960s and early 1970s and continues with a summary of the recent literature investigating normal and tumor tissue responses in electron, photon and proton beams. The section is concluded with possible mechanistic explanations of the FLASH normal-tissue protection effect (FLASH effect). Finally, challenges associated with clinical translation of FLASH-RT and its future prospects are critically discussed; specifically, proposed treatment machines and publications on treatment planning for FLASH-RT are reviewed.

The Protective Effect of Estrogen Against Radiation Cataractogenesis is Dependent Upon the Type of Radiation.

Astronauts participating in prolonged space missions constitute a population of individuals who are at an increased risk for cataractogenesis due to exposure to densely ionizing charged particles. Using a rat model, we have previously shown that after irradiation of eyes with either low-linear energy transfer (LET) 60Co γ rays or high-LET 56Fe particles, the rate of progression of anterior and posterior subcapsular cataracts was significantly greater in ovariectomized females implanted with 17-ß-estradiol (E2) compared to ovariectomized or intact rats. However, our additional low-LET studies indicated that cataractogenesis may be a modifiable late effect, since we have shown that the modulation of cataractogenesis is dependent upon the timing of administration of E2. Interestingly, we found that E2 protected against cataractogenesis induced by low-LET radiation, but only if administered after the exposure; if administered prior to and after irradiation, for the entire period of observation, then E2 enhanced progression and incidence of cataracts. Since most radioprotectors tested to date are unsuccessful in protecting against the effects of high-LET radiation, we wished to determine whether the protection mediated by E2 against radiation cataractogenesis induced by low-LET radiation would also be observed after high-LET irradiation. Female 56-day-old Sprague-Dawley rats were treated with E2 at various times relative to the time of single-eye irradiation with 2 Gy of 56Fe ions. We found that administration of E2 before irradiation and throughout the lifetime of the rat enhanced cataractogenesis compared to ovariectomized animals. The enhancing effect was slightly reduced when estrogen was removed after irradiation. However, in contrast to what we observed after γ-ray irradiation, there was no inhibition of cataractogenesis if E2 was administered only after 56Fe-ion irradiation. We conclude that protection against cataractogenesis by estrogen is dependent upon the type and ionization density of radiation that the lens was exposed to. The lack of inhibition of radiation cataractogenesis in rats that receive E2 treatment after high-LET irradiation may be attributed to the qualitative differences in the types of DNA damage induced with high-LET radiation compared to low-LET radiation or how damage may be modified at the DNA or tissue level after irradiation.

Knockdown of the DNA repair and redox signaling protein Ape1/Ref-1 blocks ovarian cancer cell and tumor growth.

Apurinic endonuclease 1/redox effector factor-1 (Ape1/Ref-1 or Ape1) is an essential protein with two distinct functions. It is a DNA repair enzyme in the base excision repair (BER) pathway and a reduction-oxidation (redox) signaling factor maintaining transcription factors in an active reduced state. Our laboratory previously demonstrated that Ape1 is overexpressed in ovarian cancer and potentially contributes to resistance. Therefore, we utilized siRNA technology to knockdown protein levels of Ape1 in ovarian cancer cell line, SKOV-3x. Knocking Ape1 down had dramatic effects on cell growth in vitro but was not due to an increase in apoptosis and at least partially due to an extension in transit time through S-phase. Similarly, human ovarian tumor xenografts with reduced levels of Ape1 protein demonstrated a dramatic reduction in tumor volume (p<0.01) and also statistically significant (p=0.02) differences in (18)F-fluorodeoxyglucose (FDG) uptake indicating reduced glucose metabolism and cellular proliferation. Ape1's role in DNA repair and redox signaling is important to our basic understanding of ovarian cancer cell growth and these findings strongly support Ape1 as a therapeutic target.

Suppression of NF-kappaB activity by parthenolide induces X-ray sensitivity through inhibition of split-dose repair in TP53 null prostate cancer cells.

We have shown that parthenolide, a sesquiterpene lactone, is a radiation sensitizer for human CGL1 hybrid cells that have constitutively activated NF-kappaB and wild-type p53. Since many malignant cells have nonfunctional p53, we investigated whether parthenolide could alter the X-ray sensitivity of PC-3 prostate cancer cells, a p53 null cell line with constitutively activated NF-kappaB. The addition of 5 microM parthenolide induced non-apoptotic cell death, inhibited PC-3 proliferation, and increased the population doubling time from 23+/-1 h to 49+/-4 h. Parthenolide also inhibited constitutive and radiation-induced NF-kappaB binding activity and enhanced the X-ray sensitivity of these p53 null PC-3 cells by a dose modification factor of 1.7. Cell cycle analysis of PC-3 cells treated with parthenolide showed only small alterations in G1 and G2/M cells, and these appeared to be insufficient to explain the observed radiosensitization. Split-dose studies using clinically relevant 2- and 4-Gy fractions demonstrated that parthenolide completely inhibited split-dose repair in PC-3 cells. We hypothesized that inhibition of NF-kappaB activity by parthenolide was responsible for the observed X-ray sensitization and inhibition of split-dose repair. To test this hypothesis, we knocked down the expression of NF-kappaB p65 protein, an active component of NF-kappaB in both PC-3 and CGL1 cells, by siRNA. Inhibition of NF-kappaB activity by knockdown of p65 increased radiation sensitivity and completely inhibited split-dose repair in both cell lines in a nearly identical manner as parthenolide treatment alone. Treating p65-depleted PC-3 cells with 5 microM parthenolide did not further increase their radiation sensitivity or the inhibition of split-dose repair. We propose that the suppression of radiation-induced NF-kappaB activity by parthenolide leads to X-ray sensitization through inhibition of split-dose repair in p53 null PC-3 prostate cancer cells.

Effect of gender on radiation-induced cataractogenesis.

Radiation cataractogenesis is an important consideration for radiotherapy patients and for astronauts. Data in the literature suggest that gender and/or estrogen may play a role in the incidence of age-related cataracts. However, few data exist on the effect of gender on radiation-induced cataractogenesis. We compared the incidence and rate of progression of cataracts induced by ionizing radiation in male and female Sprague-Dawley rats. Male rats were implanted with either an empty silastic capsule or a capsule containing 17-beta-estradiol. Ovary-intact female rats were implanted with empty capsules. All rats received a single dose of 10 Gy (60Co gamma rays) to the right eye only. Lens opacification was measured at 2-4-week intervals with a slit lamp. The incidence of radiation-induced cataracts was significantly increased in male rats compared to female rats (P=0.034). There was no difference in the rate of cataract progression between the three groups. Our data suggest there is a gender-related difference in radiation-induced cataractogenesis, but the increased incidence of radiation cataractogenesis in male rats compared to female rats cannot be attributed to estrogen levels, since there was no difference in cataract incidence between male rats implanted with empty capsules and those implanted with capsules containing 17-beta-estradiol.

Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer.

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

Reduced apoptosis and increased deletion mutations at Aprt locus in vivo in mice exposed to repeated ionizing radiation.

Exposure to ionizing radiation (IR) is a risk factor for carcinogenesis because it is a mutagen. However, a single 4-Gy whole body X-ray exposure only induced a modest increase of mutations at the Aprt reporter gene locus in mouse T cells. Intriguingly, when the same dose of IR was given in a fractionated protocol (1 Gy x 4 at weekly intervals), there was a strong induction of Aprt mutations in T cells. Many of these were mutations that arose via interstitial deletions inclusive of Aprt or by intragenic deletions. We hypothesized that the weekly fractionated X-ray exposures select for somatic cells with reduced p53 expression and/or reduced apoptosis, which, in turn, may have facilitated the accumulation of interstitial deletions, as in p53-deficient mice. We indeed found that splenocytes of mice with three previous exposures (1 Gy x 4 in total) were more resistant to X-ray-induced apoptosis than those of mice exposed to X-rays for the first time (1 Gy total). Thus, repeated X-ray radiation selects for reduced apoptosis in vivo. However, this reduced apoptosis is p53-independent, because p53 induction and the up-regulation of genes downstream of p53, such as Bax and p21, were similar between the 1-Gy and 1 Gy x 4 groups. Reduced apoptosis probably allows the generation of more mutations, particularly deletion mutations. Because both reduced apoptosis and increased somatic mutation are risk factors for carcinogenesis, they may contribute to the paradigm in which different radiation exposure schemes are varied in their efficiency in inducing lymphomagenesis.

Transcriptomic profiling of gamma ray induced mutants from the CGL1 human hybrid cell system reveals novel insights into the mechanisms of radiation-induced carcinogenesis.

BACKGROUND: Somatic cell hybrid systems generated by combining cancerous with non-cancerous cells provide useful model systems to study neoplastic transformation. Combined with recent advances in omics-based technologies, novel molecular signatures that drive radiation-induced carcinogenesis can be analyzed at an exceptional global level. METHODS: Here, we present a complete whole-transcriptome analysis of gamma-induced mutants (GIM) and gamma irradiated control (CON) segregants isolated from the CGL1 (HeLa x normal fibroblast) human hybrid cell-system exposed to high doses of radiation. Using the Human Transcriptome Array 2.0 microarray technology and conservative discrimination parameters, we have elucidated 1067 differentially expressed genes (DEGs) between tumorigenic and non-tumorigenic cells. RESULTS: Gene ontology enrichment analysis revealed that tumorigenic cells demonstrated shifts in extracellular matrix (ECM) and cellular adhesion profiles, dysregulation of cyclic AMP (cAMP) signaling, and alterations in nutrient transport and cellular energetics. Furthermore, putative upstream master regulator analysis demonstrated that loss of TGFß1 signaling due to reduced SMAD3 expression is involved in radiation-induced carcinogenesis. CONCLUSIONS: Taken together, this study presents novel insights into specific gene expression and pathway level differences that contribute to radiation-induced carcinogenesis in a human cell-based model. This global transcriptomic analysis and our published tumor suppressor gene deletion loci analyses will allow us to identify and functionally test candidate nexus upstream tumor suppressor genes that are deleted or silenced after exposure to radiation.

Analysis of the 2017 American Society for Radiation Oncology (ASTRO) Research Portfolio.

PURPOSE: Research in radiation oncology (RO) is imperative to support the discovery of new uses of radiation and improvement of current approaches to radiation delivery and to foster the continued evolution of our field. Therefore, in 2016, the American Society of Radiation Oncology performed an evaluation of research grant funding for RO. METHODS AND MATERIALS: Members of the Society of Chairs of Academic Radiation Oncology Programs (SCAROP) were asked about funded and unfunded grants that were submitted by their departments between the fiscal years 2014 and 2016. Grants were grouped according to broad categories defined by the 2017 American Society of Radiation Oncology Research Agenda. Additionally, active grants in the National Institutes of Health (NIH) Research Portfolio Online Reporting Tools database were collated using RO faculty names. RESULTS: Overall, there were 816 funded (44%) and 1031 unfunded (56%) SCAROP-reported grants. Total grant funding was over $196 million. The US government funded the plurality (42.2%; 345 of 816) of grants compared with nonprofit and industry funders. Investigators from 10 institutions accounted for >75% of funded grants. Of the funded grants, 43.5% were categorized as "genomic influences and targeted therapies." The proportion of funded to unfunded grants was highest within the category of "tumor microenvironment, normal tissue effects, and reducing toxicity" (53.4% funded). "New clinical trial design and big data" had the smallest share of SCAROP grant applications and the lowest percent funded (38.3% of grants). NIH grants to RO researchers in 2014 to 2016 accounted for $85 million in funding. From the 31 responding SCAROP institutions, there was a 28% average success rate for RO proposals submitted to the NIH during this period. CONCLUSIONS: Though RO researchers from responding institutions were relatively successful in obtaining funding, the overall amount awarded remains small. Continued advocacy on behalf of RO is needed, as well as investment to make research careers more attractive areas for emerging faculty.

X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells.

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here, we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase beta, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks, to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally regulated phenotypes, such as mitotic index.

Estrogen protects against radiation-induced cataractogenesis.

Cataractogenesis is a complication of radiotherapy when the eye is included in the treatment field. Low doses of densely ionizing space radiation may also result in an increased risk of cataracts in astronauts. We previously reported that estrogen (17-beta-estradiol), when administered to ovariectomized rats commencing 1 week before gamma irradiation of the eye and continuously thereafter, results in a significant increase in the rate and incidence of cataract formation and a decreased latent period compared to an ovariectomized control group. We therefore concluded that estrogen accelerates progression of radiation-induced opacification. We now show that estrogen, if administered continuously, but commencing after irradiation, protects against radiation cataractogenesis. Both the rate of progression and incidence of cataracts were greatly reduced in ovariectomized rats that received estrogen treatment after irradiation compared to ovariectomized rats. As in our previous study, estradiol administered 1 week prior to irradiation at the time of ovariectomy and throughout the period of observation produced an enhanced rate of cataract progression. Estrogen administered for only 1 week prior to irradiation had no effect on the rate of progression but resulted in a slight reduction in the incidence. We conclude that estrogen may enhance or protect against radiation cataractogenesis, depending on when it is administered relative to the time of irradiation, and may differentially modulate the initiation and progression phases of cataractogenesis. These data have important implications for astronauts and radiotherapy patients.

In Vitro Cell-free DNA Quantification: A Novel Method to Accurately Quantify Cell Survival after Irradiation.

Circulating tumor DNA (ctDNA) analysis has been shown to aid in both the detection of cancer and evaluation of somatic mutations in tumors. CtDNA concentration in plasma increases in proportion to tumor volume and/or metabolic activity and growth; however, this principle has yet to be applied to cell culture. We hypothesized that cell line-specific cell-free DNA (cfDNA) can be used to measure cell viability and cell survival in cell culture. Clonogenic assays on non-small cell lung cancer (NSCLC) cell lines H322, A549 and H322 were exposed to radiation doses of 0, 4 and 8 Gy. Prior to colony fixation and counting, cfDNA was extracted and quantified from cell culture media. The correlation between cell line-specific cfDNA and number of colonies grown on culture plates was examined. An H1299:A549 coculture model was used to evaluate the differential release of cell line-specific cfDNA. The results of this work indicate a strong correlation between CfDNA quantification from cell culture media and clonogenic survival at all radiation doses and in all cell lines tested (R2 range = 0.77-0.99). Cell survival curves derived from cfDNA were virtually indistinguishable from matched traditional clonogenic survival data ( P > 0.05; no significant difference exists between clonogenic curves). CfDNA quantification also accurately estimates colony count in a two-cell-line coculture model. In conclusion, cell-free DNA quantification from cell culture media can be used to measure cell survival, and appears suitable for development in a high-throughput clonogenic assay and radiosensitizer screening platform.