Ionizing radiation is carcinogenic, but genotype is a key determinant of susceptibility. Mutational DNA damage is generally attributed to cause disease, but irradiation also affects multicellular interactions as a result of poorly understood bystander effects that may influence carcinogenic susceptibility. In this study, we show that the bone marrow of irradiated mice will retain the ability to kill hemopoietic clonogenic stem cells and to induce chromosomal instability for up to 3 months after irradiation. Chromosomal instability was induced in bone marrow cells derived from CBA/Ca mice, a strain that is susceptible to radiation-induced acute myeloid leukemia (r-AML), but not in C57BL6 mice that are resistant to r-AML. Similarly, clonogenic cell lethality was exhibited in C57BL/6 mice but not CBA/Ca mice. Mechanistic investigations revealed that these genotype-dependent effects involved cytokine-mediated signaling and were mediated by a cyclooxygenase-2–dependent mechanism. Thus, our results suggested that inflammatory processes were responsible for mediating and sustaining the durable effects of ionizing radiation observed on bone marrow cells. Because most exposures to ionizing radiation are directed to only part of the body, our findings imply that genotype-directed tissue responses may be important determinants of understanding the specific consequence of radiation exposure in different individuals. Cancer Res; 71(20); 6485–91. ©2011 AACR.

Introduction: Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods: Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results: When T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both). Conclusion: Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Significance: Radiotherapy is one of the most common forms of cancer treatment used today. Its impact in directly killing cancer cells, and on antitumour immunity, is well recognized, but its effect on immune cell–cancer cell interactions is not fully understood. Here, we show that irradiation of cancer cells surprisingly leads to resistance against immune cell cytotoxicity. This was mediated by a reduced susceptibility to the pore-forming protein perforin. Resistance manifested as a reduction in both necrotic death following osmotic lysis and apoptotic death following granzyme B uptake. Overall, these data establish a form of treatment-induced resistance to lymphocyte cytotoxicity. These findings have significant implications for the optimal design of radiotherapy–immunotherapy protocols.

Radiation-induced bystander and abscopal effects, in which DNA damage is produced by inter-cellular communication, indicate mechanisms of generating damage in addition to those observed in directly irradiated cells. In this article, we show that the bone marrow of irradiated p53(+/+) mice, but not p53(-/-) mice, produces the inflammatory pro-apoptotic cytokines FasL and TNF-alpha able to induce p53-independent apoptosis in vitro in nonirradiated p53(-/-) bone marrow cells. Using a congenic sex-mismatch bone marrow transplantation protocol to generate chimeric mice, p53(-/-) hemopoietic cells functioning in a p53(+/+) bone marrow stromal microenvironment exhibited greater cell killing after irradiation than p53(-/-) hemopoietic cells in a p53(-/-) microenvironment. Cytogenetic analysis demonstrated fewer damaged p53(-/-) cells in a p53(+/+) microenvironment than p53(-/-) cells in a p53(-/-) microenvironment. Using the two different model systems, the findings implicate inflammatory tissue processes induced as a consequence of p53-dependent cellular responses to the initial radiation damage, producing cytokines that subsequently induce ongoing p53-independent apoptosis. As inactivation of the p53 tumor suppressor pathway is a common event in malignant cells developing in a stromal microenvironment that has normal p53 function, the signaling processes identified in the current investigations have potential implications for disease pathogenesis and therapy. (C) 2013 by Radiation Research Society

Ionizing radiation is unequivocally leukemogenic and carcinogenic, and this is generally attributed to DNA damage arising as a consequence of deposition of energy in the cell nucleus at the time of exposure. However, nontargeted effects, in which DNA damage is produced in nonirradiated cells as a consequence of cell signaling processes, indicate additional mechanisms. Radiation-induced chromosomal instability, a nontargeted effect with the potential to produce pathological consequences, is characterized by an increased rate of chromosome aberrations many generations after the initial insult. In this study, using a mouse model that has been well characterized with respect to its susceptibility to both radiation-induced chromosomal instability and acute myeloid leukemia, we investigated whether the underlying signaling mechanism was an inflammatory process by studying the effects of a nonsteroidal anti-inflammatory drug. Treated mice showed significant reduction in expression of the chromosomal instability phenotype 100 days postirradiation associated with reduced expression of inflammatory markers. The data support the hypothesis that the radiation-induced chromosomal instability phenotype is not an intrinsic property of the cells but a consequence of inflammatory processes having the potential to contribute secondary damage expressed as nontargeted and delayed radiation effects.