Medical countermeasures for the hematopoietic-subsyndrome of acute radiation syndrome in space.

More than 50 years after the Apollo missions ended, the National Aeronautical and Space Administration (NASA) and other international space agencies are preparing a return to the moon as a step towards deep space exploration. At doses ranging from a fraction of a Gray (Gy) to a few Gy, crew will be at risk for developing bone marrow failure associated with the hematopoietic subsyndrome of acute radiation syndrome (H-ARS) requiring pharmacological intervention to reduce risk to life and mission completion. Four medical countermeasures (MCM) in the colony stimulating factor class of drugs are now approved for treatment of myelosuppression associated with ARS. When taken in conjunction with antibiotics, fluids, antidiarrheals, antiemetics, antipyretics, and other treatments for symptomatic illness, the likelihood for recovery and mission completion can be greatly improved. The current review describes the performance and health risks of deep space flight, ionizing radiation exposure during crewed missions to the moon and Mars, and U.S. Food and Drug Administration (FDA)-approved medical interventions to treat ARS. With an expansion of human exploration missions beyond low Earth orbit (LEO), including near-term Lunar and future Mars missions, inclusion of MCMs to counteract ARS in the spaceflight kit will be critical for preserving crew health and performance.

Secondary Malignancies in the Era of High-Precision Radiation Therapy.

Although modern radiation therapy delivers a localized distribution of ionizing energy that can be used to cure primary cancers for many patients, the inevitable radiation exposure to non-targeted normal tissue leads to a risk of a radiation-related new cancer. Modern therapies often produce a complex spectrum of secondary particles, both charged and uncharged, that must be considered both in their physical radiation transport throughout the patient and their potential to induce biological damage, which depends on the microscopic energy deposition from the cascade of primary, secondary, and downstream particles. This work summarizes the experimental data for relative biological effectiveness for particles associated with modern radiotherapy in light of their capacity to induce secondary malignancies in patients. A distinction is highlighted between the radiobiological experimental data and the coarser metrics used frequently in radiation protection. For critical assessment of the risks of secondary malignancies for patients undergoing radiation therapy, a detailed description of primary and secondary radiation fields is needed, though not routinely considered for individual patient treatments. Furthermore, not only the particle type, but also the microscopic dose and track structure, must be considered, which points to a demand for detailed physics models and high-performance computing strategies to model the risks.

The Role of the Apoptotic Machinery in Ionizing Radiation-Induced Carcinogenesis.

Ionizing radiation is an established cause of cancer based on epidemiologic and experimental evidence. According to epidemiological data, virtually all tissues in the human body are sensitive to the carcinogenic action of radiation. Apoptosis represents a major barrier to cancer because apoptotic cell death eliminates dangerous cells that may initiate tumor development. The explosion in research on apoptosis in the 1990s has demonstrated that irradiated cells have differential apoptotic proclivities and has suggested that radiation can affect apoptosis by inducing the type of damage that is eliminated by programmed cell death but may also modulate the efficiency of this damage removal process. The purposes of this review are to summarize some earlier studies on carcinogenesis that serve as a reminder of the background on which more recent studies are based, to highlight examples of progress in cancer and apoptosis research in the context of radiation carcinogenesis, to indicate gaps in our knowledge, and to point out possibly fertile directions for future investigation.

Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon.

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation. These RAD mutants displayed higher transcription levels for the rfa3 operon, coding two subunits of the RPA-like putative single-stranded binding protein, rfa3 and rfa8, and a third downstream gene, ral. In order to bioengineer cells with increased tolerance to ionizing radiation and further explore the genetic basis of the RAD phenotype, we placed the rfa3 operon under control of the gvpA promoter in a Halobacterium expression plasmid, pDRK1. When pDRK1 was introduced into the wild-type NRC-1 strain, overproduction of the Rfa3 and Rfa8 proteins was observed by Western blotting and proteomic analysis. The Halobacterium strains expressing Rfa3 and Rfa8 also displayed improved survival after exposure to ionizing radiation, similar to the RAD mutants, when compared to wild-type strain NRC-1. The Rfa3 and Rfa8 proteins co-purified by affinity chromatography on single-stranded DNA cellulose columns, confirming the ability of the proteins to bind to single-stranded DNA as well as their relative abundance in the wild-type, RAD mutants, and rfa3 operon overexpression strains. These results clearly establish that overexpression of haloarchaeal RPA promotes ionizing radiation resistance in Halobacterium sp. NRC-1 and that the Rfa3 and Rfa8 subunits bind single-stranded DNA. Bioengineering cells with increased levels of ionizing radiation resistance may have potential value in medical and environmental applications.

The effect of docetaxel (taxotere) on human gastric cancer cells exhibiting low-dose radiation hypersensitivity.

Low-dose radiation hypersensitivity (HRS) describes a phenomenon of excessive sensitivity to X ray doses <0.5 Gy. Docetaxel is a taxane shown to arrest cells in the G(2)/M phase of the cell cycle. Some previous studies suggested that HRS might result from the abrogation of the early G(2) checkpoint arrest. First we tested whether HRS occurs in gastric cancer-derived cells, and whether pre-treatment of cells with low docetaxel concentrations can enhance the magnitude of HRS in gastric cancer cells. The results demonstrated HRS at ~0.3 Gy and the synergy between 0.3 Gy and docetaxel (3 nM for 24 h), and the additivity of other drug/dose combinations. The synergistic effect was associated with a significant docetaxel-induced G(2) accumulation. Next, we evaluated in time-course experiments ATM kinase activity and proteins associated with the induction and maintenance of the early G(2) checkpoint. The results of multi-immunoblot analysis demonstrate that HRS does not correlate with the ATM-dependent early G(2) checkpoint arrest. We speculate that G(2) checkpoint adaptation, a phenomenon associated with a prolonged cell cycle arrest, might be involved in HRS. Our results also suggest a new approach for the improvement the effectiveness of docetaxel-based radiotherapy using low doses per fraction.

The synergistic effect of dimethylamino benzoylphenylurea (NSC #639829) and X-irradiation on human lung carcinoma cell lines.

PURPOSE: The present study was designed to investigate the ability of N-[4-(5-bromo-2-pyrimidyloxy)-3-methylphenyl]-(dimemethylamino)-benzoylphenylurea (dimemethylamino benzoylphenylurea; BPU) to sensitize cells to radiation and to examine the relationship between phenotype versus survival, DNA damage, apoptosis, or cell cycle progression in non-small cell lung cancer (NSCLC) cell lines. METHODS: Asynchronous cultures of three NSCLC (phenotype) lines, A549 (adenocarcinoma), NCI-H226 (squamous) and NCI-H596 (adenosquamous) were used. Cells were treated for 24 h with BPU at various concentrations (0-10 microM) to obtain drug doses for inhibiting cell survival by approximately 50% (IC50). Cells were X-irradiated without BPU or after 24 h BPU treatment at IC50. Radiation doses ranged from 0 to10 Gy. Cell survival was determined by a colony-forming ability assay. The effect of BPU on the cell cycle distribution and induction of apoptosis were measured by flow cytometry-based assays. The effect of BPU on radiation-induced DNA damage and repair was analyzed according to nuclear gammaH2AX immunofluorescence of cells exposed to X-rays alone or after BPU. Anti-gammaH2AX antibody staining, a surrogate determinant of double stranded DNA breaks, was measured using flow cytometry. RESULTS: BPU (1.5 microM) for 24 h produced approximately 50% cell survival. BPU and X-irradiation were synergistic in the three cell lines at survival levels of 20-50%. Flow cytometry analysis of replicate experiments with BPU (1.5 microM for 24 h) showed that BPU blocked cell progression at S and/or G2/M. The incidence of apoptosis in BPU-treated versus control cells ranged from approximately 0.3 to approximately 8%. Twenty-four hour after X-irradiation cells pre-treated with BPU and X-irradiated after drug exposure showed gammaH2AX levels approximately two times higher than did the cells exposed to X-rays only. CONCLUSIONS: The study identified BPU as a novel radiation sensitizer. The analysis of phosphorylated histone H2AX as a surrogate marker of DNA double strand breaks suggested a positive association between radiosensitization and the inhibition of X-irradiation-induced DNA damage repair by BPU.

Cytotoxicity of docetaxel (Taxotere) used as a single agent and in combination with radiation in human gastric, cervical and pancreatic cancer cells.

BACKGROUND: Docetaxel (Taxotere) has gained increasing attention in clinical applications. We investigated the cytotoxic and radiosensitizing potential of docetaxel at nanomolar concentrations in six cell lines derived from tumors that rarely respond to radiation or chemotherapy, with special consideration of mechanisms of resistance, including the p53 mutational status. METHODS: Cells derived from carcinomas of the human stomach (p53 mutant Hs746T, p53 wild type AGS), cervix (p53 wild type CaSki, p53 mutant HeLa) or pancreas (p53 mutant BxPC3 and Capan-1) were treated for 24 h with docetaxel at various concentrations (0.1-5 nM) to obtain drug doses for inhibiting clonogenicity by approximately 50% (IC(50)). Cells were X-irradiated without docetaxel or after 24 h of docetaxel treatment at IC(50). Radiation doses ranged from 0 up to 10 Gy. Mitotic index, multinucleation, apoptosis and necrosis after 24 h of drug exposure at 1 nM were quantified in representative gastric and cervical cell lines by fluorescence microscopy. RESULTS: Docetaxel treatment for 24 h resulted in a dose-dependent loss of clonogenicity, with 1.0 or 0.3 nM producing approximately 50% survival of gastric or cervix and pancreatic cells, respectively. After correction for the drug toxicity, the combination of isoeffective concentrations of docetaxel with graded X-ray doses resulted either in a moderate synergy or additivity. The dose reduction factors at the 50 and 20% survival levels were statistically greater than those for Hs746T or AGS cells. For CaSki, HeLa, BxPC3 or Capan-1 cells, the dose reduction factors were statistically not different from unity. CONCLUSION: Docetaxel was active against tumor cells of different origins. Combined effects of docetaxel and radiation were at least additive and depended on the intrinsic sensitivity to drug alone. There was no significant evidence of drug-induced mitotic arrest. Compared to drug-resistant gastric cells, exposure to the drug alone of drug-sensitive cervical cells resulted in more severe multinucleation. The p53 status did not contribute directly to the effect of drug alone or in combination with radiation.

Coordinate late expression of trefoil peptide genes (pS2/TFF1 and ITF/TFF3) in human breast, colon, and gastric tumor cells exposed to X-rays.

The trefoil factors (TFFs) are pleiotropic factors involved in organization and homeostasis of the gastrointestinal tract, estrogen responsiveness, inflammatory disorders, and carcinogenesis. In an earlier study using cDNA array technologies to identify new genes expressed in irradiated cell survivors, we isolated a cDNA clone corresponding to the reported human TFF1 gene (E. K. Balcer-Kubiczek et al., Int. J. Radiat. Biol., 75: 529-541, 1999). To determine whether expression of other TFFs is altered by ionizing radiation, we quantified changes in expression of TFF3 as well as TFF1 in RNA samples obtained from irradiated and control human tumor breast, colon, and gastric tumor cells and examined expression kinetics up to 2 weeks after irradiation. X-ray-induced TFF1 and TFF3 expression profiles were compared with those induced by hydrogen peroxide (H2O2) or 17beta-estradiol (ES). The results revealed that TFF1 and TFF3 mRNA are coinduced by X-irradiation in a subset of the lines, but substantial heterogeneity in their responses was observed in cells derived from a single cell type. TFF1 and TFF3 transcriptional response to X-irradiation differed from that to H2O2 or ES in the timing of their induction as well as tissue-type dependence, i.e., their induction pattern after X-irradiation was late and sustained, whereas their induction by H2O2 or ES was early and transient. TFF1 mRNA, protein production in the cytoplasm, and secretion in the culture supernatant were coordinately regulated after X-irradiation. There was no requirement for TP53 in this induction. These results demonstrate the existence of a novel class of radiation-responsive genes that might be involved in bystander effects.