The effect of radiation on the ability of rat mammary cells to form mammospheres

As classical transplantation repopulation assays for studying the radiobiology of rat mammary stem/progenitor cells are extremely time-consuming, this study aimed to characterize the radiobiological properties of mammospheres, spherical clumps of mammary cells formed under non-adherent culture conditions, which are a simple and widely used technique for assessing progenitor cell activity. Rat mammary cells were dissociated and used in transplantation repopulation assays and for the formation of mammospheres. Immunofluorescence for cytokeratin 14 and 18 was used to identify basal and luminal mammary epithelial cells, respectively. Incorporation of 5-bromo-2’-deoxyuridine (BrdU) was used to evaluate cell proliferation. The repopulating activity of the transplanted primary rat mammary cells demonstrated their radiosensitivity, reproducing previous data, with a significant reduction in repopulating activity at ≥2 Gy. Cells constituting rat mammospheres were positive for either cytokeratin 14 or 18, with occasional double-positive cells. Both proliferation and aggregation contributed to sphere formation. Cells obtained from the spheres showed lower repopulating activity after transplantation than primary cells. When primary cells were irradiated and then used for sphere formation, the efficiency of sphere formation was significantly decreased at 8 Gy but not at ≤6 Gy, indicating radioresistance of the formation process. Irradiation at 8 Gy reduced the proliferation of cells during sphere formation, whereas the cellular composition of the resulting spheres was unaffected. Thus, mammosphere formation assays may measure a property of putative mammary progenitors that is different from what is measured in the classic transplantation repopulation assay in radiobiology

Radiobiology of sphere-forming rat mammary epithelial cells under nonadherent culture

The mammary gland is highly susceptible to radiation carcinogenesis. It is vital to delineate the effect of radiation on tissue stem and progenitor cells, from which cancer cells arise, to understand the carcinogenic mechanisms of radiation. Herein we established a technique for the formation of ‘mammospheres’ of rat mammary epithelial cells (RMECs) using the nonadherent culturing method, which was previously developed for culturing human mammary progenitor cells, and examined the biological and radiobiological characteristics of mammosphere-forming cells. We found that mammospheres mainly consisted of proliferating cells that were double positive for cytokeratin (CK)14 and CK18 (markers for myoepithelial and luminal epithelial cells, respectively). Under differentiating conditions in adherent culture, mammospheres generated both CK14 and CK18 single-positive cells, implying that mammospheres consist of bipotent progenitor cells. Orthotropic transplantation assays showed that mammospheres contained lower stem cell activity than primary RMECs, suggesting that mammospheres are not enriched in stem cells. When primary RMECs were irradiated with up to 8 Gy in suspension and subjected to mammosphere formation, the efficiency of mammosphere formation, bromodeoxyuridine incorporation of cells therein, CK14/CK18 expression in mammospheres and their generation of single-positive cells in adherent culture were unaffected. These results suggest that mammosphere-forming cells of RMECs are resistant to radiation-induced proliferative death and perturbation of differentiation.第15回幹細胞シンポジウ

Prominent Dose-Rate Effect and Its Age Dependence of Rat Mammary Carcinogenesis Induced by Continuous Gamma-Ray Exposure

Although the risk of breast cancer after high dose rate radiation exposure has been firmly established, the risk incurred by low dose rates is not well understood. Herein, we provide experimental evidence for dose rate and age dependencies induced by continuous γ-ray irradiation on mammary carcinogenesis. Female rats were subjected to continuous whole-body irradiation i) at 7 weeks of age (denoted adults) at a dose rate of 360 mGy/h (4 Gy total) or ii) at 3 weeks of age (denoted juveniles) or as 7-week-old adults at a dose rate of 6 mGy/h (18 Gy total). Additional rats were acutely irradiated at 13 weeks of age at a dose rate of 30 Gy/h (0.5–4 Gy total). We observed the incidence of mammary tumors by weekly palpation until they were 90 weeks old and following pathological inspection upon autopsy. The tumor incidence rate for each group was characterized by Cox regression analysis. When adult rats were irradiated at 60 mGy/h for a total of 4 Gy, their hazard ratio for mammary carcinoma significantly increased relative to unirradiated controls; however, for adult rats irradiated at 3–24 mGy/h, even though they also received a total of 4 Gy, their hazard ratio for carcinoma incidence did not significantly increase. A larger increase in the incidence rate of carcinoma per dose was found for the juveniles than for the adults irradiated at 6 mGy/h, whereas age did not influence the effect of acute irradiation at 30 Gy/h; a threshold-like dose response was observed for irradiation at 6 mGy/h (threshold, ~2.5 and ~4 Gy for juveniles and adults, respectively). Regarding benign tumors of the mammary gland, a significant increase in their incidence was observed for irradiation down to 6 mGy/h, but not at 3 mGy/h, and there was no evidence of age-dependent induction. Thus, induction of female rat mammary carcinogenesis by continuous γ-ray irradiation was age dependent and drastically increased for adult rats between 24 and 60 mGy/h

Age Modifies the Effect of 2-MeV Fast Neutrons on Rat Mammary Carcinogenesis

The relative biological effectiveness (RBE) of neutrons depends on their physical nature (e.g., energy) and the biological context (e.g., endpoints, materials). From the perspective of radiological protection, age is an important biological context that influences radiation-related cancer risk, but very few studies have addressed its potential impact on neutron effects. We therefore investigated the influence of age on the effect of accelerator-generated fast neutrons (mean energy, ~2 MeV) in an animal model of breast carcinogenesis. Female Sprague-Dawley rats at 1, 3, and 7 weeks of age were irradiated with fast neutrons at absorbed doses of 0.0485–0.97 Gy. All animals were kept under specific pathogen–free conditions and screened weekly for mammary tumors by palpation until they were 90 weeks old. Tumors were diagnosed based on histology. Mathematical modeling was used to analyze mammary cancer incidence, collectively using data from this study and a previously reported experiment on 137Cs γ rays. The results indicate that neutron irradiation elevated the risk of palpable mammary carcinoma with a linear dose response, the slope of which depended on age at the time of irradiation. The RBE of neutron irradiation was 7.5 ± 3.4, 9.3 ± 3.5, and 26.1 ± 8.9 (mean ± SE) for animals treated at 1, 3, and 7 weeks of age, respectively. Our results indicate that animal age is an important factor influencing the effect of fast neutrons on breast cancer risk

Biological measures to minimize the risk of radiotherapy-associated second cancer: a research perspective

Purpose: Second cancers are among the most serious sequelae for cancer survivors who receive radiotherapy. This article aims to review current knowledge regarding how the risk of radiotherapy-associated second cancer can be minimized by biological measures and to discuss relevant research needs. Results: The risk of second cancer can be reduced not only by physical measures to decrease the radiation dose to normal tissues but also by biological means that interfere with the critical determinants of radiation-induced carcinogenesis. Requirements for such biological means include the targeting of tumor types relevant to radiotherapy-associated risk, concrete safety and efficacy evidence and feasibility and minimal invasiveness. Mechanistic insights into the process of radiation carcinogenesis provide rational approaches to minimize the risk. Five mechanism-based strategies are proposed herein based on the current state of knowledge. Epidemiological studies on the joint effects of radiation and lifestyle or other factors can provide evidence for factors that modify radiation-associated risks if deliberately controlled.Conclusions: Mechanistic and epidemiological evidence indicates that it is possible to develop interventional measures to minimize the second cancer risk associated with radiotherapy. Research is needed regarding the critical determinants of radiation-induced carcinogenesis available for intervention and joint effects of radiation and controllable factors