Vive la radiorésistance!: converging research in radiobiology and biogerontology to enhance human radioresistance for deep space exploration and colonization.

While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments and the high costs associated with protection from the deleterious physiological effects of exposure to high-Linear energy transfer (high-LET) radiation. Herein, we lay the foundations of a roadmap toward enhancing human radioresistance for the purposes of deep space colonization and exploration. We outline future research directions toward the goal of enhancing human radioresistance, including upregulation of endogenous repair and radioprotective mechanisms, possible leeways into gene therapy in order to enhance radioresistance via the translation of exogenous and engineered DNA repair and radioprotective mechanisms, the substitution of organic molecules with fortified isoforms, and methods of slowing metabolic activity while preserving cognitive function. We conclude by presenting the known associations between radioresistance and longevity, and articulating the position that enhancing human radioresistance is likely to extend the healthspan of human spacefarers as well

The Molecular and Cellular Strategies of Glioblastoma and Non-Small-Cell Lung Cancer Cells Conferring Radioresistance

Ionizing radiation (IR) has been shown to play a crucial role in the treatment of glioblastoma (GBM; grade IV) and non-small-cell lung cancer (NSCLC). Nevertheless, recent studies have indicated that radiotherapy can offer only palliation owing to the radioresistance of GBM and NSCLC. Therefore, delineating the major radioresistance mechanisms may provide novel therapeutic approaches to sensitize these diseases to IR and improve patient outcomes. This review provides insights into the molecular and cellular mechanisms underlying GBM and NSCLC radioresistance, where it sheds light on the role played by cancer stem cells (CSCs), as well as discusses comprehensively how the cellular dormancy/non-proliferating state and polyploidy impact on their survival and relapse post-IR exposure

Composition of Conditioned Media from Radioresistant and Chemoresistant Cancer Cells Reveals miRNA and Other Secretory Factors Implicated in the Development of Resistance

Resistance to chemo- or radiotherapy is the main obstacle to consistent treatment outcomes in oncology patients. A deeper understanding of the mechanisms driving the development of resistance is required. This review focuses on secretory factors derived from chemo- and radioresistant cancer cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and cancer stem cells (CSCs) that mediate the development of resistance in unexposed cells. The first line of evidence considers the experiments with conditioned media (CM) from chemo- and radioresistant cells, CAFs, MSCs, and CSCs that elevate resistance upon the ionizing radiation or anti-cancer drug exposure of previously untreated cells. The composition of CM revealed factors such as circular RNAs; interleukins; plasminogen activator inhibitor; and oncosome-shuttled lncRNAs, mRNAs, and miRNAs that aid in cellular communication and transmit signals inducing the chemo- and radioresistance of sensitive cancer cells. Data, demonstrating that radioresistant cancer cells become resistant to anti-neoplastic drug exposure and vice versa, are also discussed. The mechanisms driving the development of cross-resistance between chemotherapy and radiotherapy are highlighted. The secretion of resistance-mediating factors to intercellular fluid and blood brings attention to its diagnostic potential. Highly stable serum miRNA candidates were proposed by several studies as prognostic markers of radioresistance; however, clinical studies are needed to validate their utility. The ability to predict a treatment response with the help of the miRNA resistance status database will help with the selection of an effective therapeutic strategy. The possibility of miRNA-based therapy is currently being investigated with ongoing clinical studies, and such approaches can be used to alleviate resistance in oncology patients

Long-Term Cultured Human Glioblastoma Multiforme Cells Demonstrate Increased Radiosensitivity and Senescence-Associated Secretory Phenotype in Response to Irradiation

The overall effect of senescence on cancer progression and cancer cell resistance to X-ray radiation (IR) is still not fully understood and remains controversial. How to induce tumor cell senescence and which senescent cell characteristics will ensure the safest therapeutic strategy for cancer treatment are under extensive investigation. While the evidence for passage number-related effects on malignant primary cells or cell lines is compelling, much less is known about how the changes affect safety and Senescence-Associated Secretory Phenotype (SASP), both of which are needed for the senescence cell-based vaccine to be effective against cancer. The present study aimed to investigate the effects of repeated passaging on the biological (self-renewal capacity and radioresistance) and functional (senescence) characteristics of the different populations of short- and long-term passaging glioblastoma multiforme (GBM) cells responding to senescence-inducing DNA-damaging IR stress. For this purpose, we compared radiobiological effects of X-ray exposure on two isogenic human U87 cell lines: U87L, minimally cultured cells (<15 passages after obtaining from the ATCC) and U87H, long-term cultured cells (>3 years of continuous culturing after obtaining from the ATCC). U87L cells displayed IR dose-related changes in the signs of IR stress-induced premature senescence. These included an increase in the proportion of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, and concomitant decrease in the proportion of Ki67-positive cells and metabolically active cells. However, reproductive survival of irradiated short-term cultured U87L cells was higher compared to long-term cultured U87H cells, as the clonogenic activity results demonstrated. In contrast, the irradiated long-term cultured U87H cells possessed dose-related increases in the proportion of multinucleated giant cancer cells (MGCCs), while demonstrating higher radiosensitivity (lower self-renewal) and a significantly reduced fraction of DNA-replicating cells compared to short-term cultured U87L cells. Conditioned culture medium from U87H cells induced a significant rise of SA-β-Gal staining in U87L cells in a paracrine manner suggesting inherent SASP. Our data suggested that low-dose irradiated long-term cultured GBM cells might be a safer candidate for a recently proposed senescence cell-based vaccine against cancer

Dose-Dependent Shift in Relative Contribution of Homologous Recombination to DNA Repair after Low-LET Ionizing Radiation Exposure: Empirical Evidence and Numerical Simulation

Understanding the relative contributions of different repair pathways to radiation-induced DNA damage responses remains a challenging issue in terms of studying the radiation injury endpoints. The comparative manifestation of homologous recombination (HR) after irradiation with different doses greatly determines the overall effectiveness of recovery in a dividing cell after irradiation, since HR is an error-free mechanism intended to perform the repair of DNA double-strand breaks (DSB) during S/G2 phases of the cell cycle. In this article, we present experimentally observed evidence of dose-dependent shifts in the relative contributions of HR in human fibroblasts after X-ray exposure at doses in the range 20–1000 mGy, which is also supported by quantitative modeling of DNA DSB repair. Our findings indicate that the increase in the radiation dose leads to a dose-dependent decrease in the relative contribution of HR in the entire repair process

Long-Term Cultured Human Glioblastoma Multiforme Cells Demonstrate Increased Radiosensitivity and Senescence-Associated Secretory Phenotype in Response to Irradiation

The overall effect of senescence on cancer progression and cancer cell resistance to X-ray radiation (IR) is still not fully understood and remains controversial. How to induce tumor cell senescence and which senescent cell characteristics will ensure the safest therapeutic strategy for cancer treatment are under extensive investigation. While the evidence for passage number-related effects on malignant primary cells or cell lines is compelling, much less is known about how the changes affect safety and Senescence-Associated Secretory Phenotype (SASP), both of which are needed for the senescence cell-based vaccine to be effective against cancer. The present study aimed to investigate the effects of repeated passaging on the biological (self-renewal capacity and radioresistance) and functional (senescence) characteristics of the different populations of short- and long-term passaging glioblastoma multiforme (GBM) cells responding to senescence-inducing DNA-damaging IR stress. For this purpose, we compared radiobiological effects of X-ray exposure on two isogenic human U87 cell lines: U87L, minimally cultured cells (3 years of continuous culturing after obtaining from the ATCC). U87L cells displayed IR dose-related changes in the signs of IR stress-induced premature senescence. These included an increase in the proportion of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, and concomitant decrease in the proportion of Ki67-positive cells and metabolically active cells. However, reproductive survival of irradiated short-term cultured U87L cells was higher compared to long-term cultured U87H cells, as the clonogenic activity results demonstrated. In contrast, the irradiated long-term cultured U87H cells possessed dose-related increases in the proportion of multinucleated giant cancer cells (MGCCs), while demonstrating higher radiosensitivity (lower self-renewal) and a significantly reduced fraction of DNA-replicating cells compared to short-term cultured U87L cells. Conditioned culture medium from U87H cells induced a significant rise of SA-β-Gal staining in U87L cells in a paracrine manner suggesting inherent SASP. Our data suggested that low-dose irradiated long-term cultured GBM cells might be a safer candidate for a recently proposed senescence cell-based vaccine against cancer

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 1011 W∙cm−2 peak power density. An increase in the pulse power density to 2 × 1011 W∙cm−2 led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 1011 W∙cm−2 led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2–6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses

DNA Comet Giemsa Staining for Conventional Bright-Field Microscopy

This study was undertaken to evaluate the compatibility of Giemsa staining protocol with the comet assay. We showed, for the first time, that DNA comets can be visualized and analyzed using Giemsa staining. We generated DNA damage dose response curves for human peripheral blood lymphocytes exposed to X-ray radiation using the comet assay with either SybrGreen I or Giemsa stain. The dose response curves were fitted by linear regressions (R2 > 0.977). The SybrGreen I results showed only ~1.2-fold higher slope coefficient (method sensitivity) compared to the Giemsa results. The unexpectedly high sensitivity of Giemsa staining for the comet assay is due to the Romanowsky-Giemsa effect, the stain photo-stability and the higher resolution of bright-field imaging compared to fluorescence imaging. Our results demonstrate that Giemsa staining can effectively be used for measuring DNA damage by the comet assay. The low cost and availability of Giemsa stain makes this method affordable for any low budget research and will facilitate new applications of the comet assay in biology and medicine

IR-Surviving NSCLC Cells Exhibit Different Patterns of Molecular and Cellular Reactions Relating to the Multifraction Irradiation Regimen and p53-Family Proteins Expression

Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins