Cellular responses and gene expression profiles of colonic Lgr5+ stem cells after low-dose/low-dose-rate radiation exposure.

We previously found that high-dose-rate radiation induced a replenishment of the colonic Lgr5+ stem cell pool, whereas low-dose-rate radiation did not. To identify key molecules that determine the dose-rate effects on this stem cell pool, we harvested colonic Lgr5+ stem cells by cell sorting at 2 weeks after exposure to 1 Gy of high-dose-rate (30 Gy/h) or low-dose-rate (0.003 Gy/h) radiation and analyzed their gene expression profiles using RNA-Seq. We found that pathways related to DNA damage response, cell growth, cell differentiation and cell death were upregulated in Lgr5+ stem cells irradiated with high dose rates, whereas pathways related to apical junctions and extracellular signaling were upregulated in low-dose-rate-irradiated colonic Lgr5+ stem cells. Interestingly, biological events involving apical junctions are known to play an important role in the exclusion of transformed cells that are surrounded by normal epithelial cells through 'cell competition'. We speculated that cell competition, through apical junctions and extracellular ligands, might contribute to the dose-rate effect on Lgr5+ cell replenishment. To understand this mechanism, we focused on 69 genes that were significantly upregulated in low-dose-rate-irradiated cells, which we named DREDGE (Dose-Rate Effect Determining GEnes). Based on these findings, we propose a possible mechanism underlying the dose-rate effect observed in the colonic stem cell pool.

INTRODUCTION

Organs and tissues comprise different types of functional cells that play specific roles. These functional cells are replaced by cellular turnover; however, the rate of replacement differs among tissues. Since functional cells have a limited lifetime, they have to be replenished by parental progenitor cells, which are continuously regenerated by stem cells. According to the ‘cell-of-origin in cancer’ model, solid tumors are considered to originate from tissue stem cells that undergo genetic mutations [1]. Ionizing radiation, a physical mutagen, induces DNA damage and genetic mutations. The number of DNA double-strand breaks (DSBs) increases in a dose-dependent manner, and insufficient repair of DSBs leads to mutations, genomic instability, and chromosomal aberrations [2-4]. Therefore, cancer is considered to originate from tissue stem cells with accumulated DNA damage [5, 6]. We hypothesized that DNA damage accumulates in pools of tissue stem cells, as they have sufficient self-renewability to maintain a tissue throughout their lifetime [7-9]. Recent studies have suggested that cancer risk in humans is related to the total numbers of stem cell divisions, and that accumulation of mutations caused by steady-state cell division does not depend on environmental factors [10, 11]. This ‘bad luck’ hypothesis is a controversial issue in stem cell research and suggests that tissue stem cells are important as a target of cancer origin.

Cancer risk induced by ionizing radiation does not seem to be determined by the cumulative dose of exposure. An epidemiological study has shown that the incidence of solid cancers among atomic bomb survivors increased with the radiation dose, exhibiting a linear dose–response relationship [12]. On the other hand, another epidemiological study reported that cancer incidence among individuals living in high-background radiation areas (HBRAs), where they receive extremely low-dose-rate radiation throughout their life, did not increase with the cumulative dose of radiation [13]. Thus, the discrepancies between these studies may be attributed to the dose rate, rather than the overall dose. ‘Dose-rate effects’ are well-known responses in which the biological effect of low-dose rate radiation is lower than that of the same dose at high-dose-rate radiation [14]. However, there is no evidence of a dose-rate effect on tissue stem cells. Therefore, we addressed this gap in knowledge to understand the biological mechanisms behind dose rate–dependent carcinogenesis.

INTESTINAL STEM CELLS AS AN ORIGIN OF CARCINOGENESIS

Intestines are the major target organ in radiation-induced cancer [15]. The intestinal epithelium consists of epithelial monolayer cells, all of which are derived from intestinal stem cells, which are located at the bottom of the crypts. Driver mutations of genes such as Apc and Ctnnb (β-catenin) of the intestinal tissue stem cells can trigger carcinogenesis [16-18]. However, for progenitors and terminally differentiated cells, driver mutations are insufficient to trigger carcinogenesis; further stimulations such as severe inflammation are required for tumor development, in addition to the acquisition of driver mutations [19]. Intestinal crypts contain stem cells with different characteristics such as actively cycling and slow cycling, which can be distinguished by their molecular markers as shown in Fig. 1 [20]. For instance, intestinal stem cells expressing leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) are cycling stem cells, which are necessary for maintaining tissue in a steady state. Lgr5 was first identified as a molecular marker on stem cells that could develop into tumors as cells of origin in cancer; for example, adenomas were induced when the Apc gene was specifically depleted in Lgr5+ stem cells [16]. Parts of both the small intestine, such as the duodenum, and the large intestine, such as the colon, contain Lgr5+ stem cells in the bottom of crypts. Besides Lgr5, markers for actively cycling stem cells such as Ascl2 and Olfm4 are also expressed in crypt base columnar (CBC) cells [21, 22]. Quiescent stem cells, which express markers such as Bmi-1 and mTert, play an important role in the repopulation of actively cycling stem cells when the pool undergoes severe damage from stress, such as high-dose radiation exposure [17, 23].

Fig. 1.

Stem cell populations in colonic crypts. Bold gene names denote common stem cell markers. Functional cells include enteroendocrine cells, goblet cells, and enterocytes.

THE DOSE-RATE EFFECT IN REPLENISHMENT OF COLONIC LGR5+ STEM CELLS

We previously found that colonic Lgr5+ stem cells were highly radiosensitive, compared with duodenal Lgr5+ stem cells, because the number of colonic Lgr5+ stem cells significantly decreased after exposure to 1 Gy of high-dose-rate (30 Gy/h) radiation [24]. As the dose-rate effect has not been evaluated in these cells, we studied the effect of radiation on Lgr5+ stem cells using the Lgr5-lineage tracing technique. This is a common technique for understanding the stem cell fate by tagging specific stem cells and their daughter cells with a reporter gene such as lacZ or a gene for a fluorescent protein, based on tamoxifen-driven Cre–loxP recombination.

In this study, we compared the effects of high-dose-rate (30 Gy/h) and low-dose-rate (0.003 Gy/h) radiation on the replenishment of Lgr5+ stem cells using Lgr5-EGFP-IRES-Cre× ROSA26-LSL-LacZ mice. In these mice, Lgr5+ stem cells constantly express Cre recombinase fused to a modified estrogen receptor (ERT2). As a ligand, tamoxifen (4-hydroxytamoxifen) binds to ERT2 and induces translocation of Cre recombinase to the nucleus, where Cre recombinase cuts out the translational stop sequence (LSL) and activates expression of the lacZ gene. A significant loss of LacZ+ crypts was observed after high-dose-rate irradiation, suggesting the replenishment of the Lgr5+ stem cell pool by quiescent stem cells [24]. However, no significant acceleration of stem cell replenishment was observed upon low-dose-rate irradiation [25]. We also studied the kinetics of DNA repair and tissue response by quantifying the number of 53BP1 foci in each cell, which is a surrogate marker for DSBs, and the number of cells expressing Ki-67 and phosphorylated histone H3 (PH3), which are markers of proliferating and mitotic cells, respectively. After high-dose rate irradiation, the number of 53BP1 foci immediately increased in colonic Lgr5+ stem cells, but DSBs were efficiently repaired thereafter. High-dose-rate radiation also induced considerable reduction in cell numbers in the colonic crypts and dramatic increase in mitosis, which may stimulate the replenishment of the stem cell pool [26]. Therefore, the abnormal growth stimulation to replenish the Lgr5+ stem cell pool may contribute to the accumulation of genetic mutations in tissue stem cells. Based on these findings, we speculated that the dose rate, rather than the cumulative dose, might contribute to the replenishment of tissue stem cells and the dose-rate effect on tissue stem cell turnover might affect cancer risk. High-dose-rate whole-body irradiation reduces the number of tissue stem cells by inducing cell death [27, 28]. Even if the cells do not die at the time of radiation, all tissue stem cells are simultaneously damaged by high-dose-rate irradiation. DNA damage can lead to aging and exhaustion of stem/progenitor cells in tissue stem cells [29, 30]. Quiescent (or slow-cycling) stem cells must rescue tissues after drastic loss of the actively cycling stem cells to repopulate the stem cell pool [31]. In fact, the proliferation of slow-cycling stem cells is induced by high-dose irradiation [23]. In the hematopoietic system, DSBs in quiescent stem cells have to be repaired by non-homologous end joining (NHEJ), although DSBs on cycling stem cells can be repaired by homologous recombination (HR), which is relatively error-free compared with NHEJ [32]. Therefore, quiescent stem cells are considered intrinsically vulnerable to genetic mutation. Growth stimulation of those quiescent stem cells after loss of cycling stem cells may trigger replenishment and expansion of mutated stem cells in the cycling stem cell pool. Thus, for tissue stem cells, preventing proliferation of slow-cycling stem cells is important for tissue integrity.

ISOLATION OF CANDIDATE GENES DETERMINING THE DOSE-RATE EFFECT

The dose-rate effect on the replenishment of Lgr5+ stem cells is a possible mechanism that circumvents the accumulation of mutated cells in its pool. To confirm our hypothesis, we identified key molecules involved and the mechanism behind the induction of the dose-rate effect, by comparing gene expression profiles in colonic Lgr5+ cells of mice exposed to high-dose-rate and low-dose-rate irradiation. We harvested colonic Lgr5+ cells by cell sorting of the EGFP+ population at 2 weeks after exposure to 1 Gy of high-dose-rate (30 Gy/h) or low-dose-rate (0.003 Gy/h) radiation. RNA-Seq was used to analyze gene expression profiles of the harvested cells. A gene set enrichment analysis revealed that pathways related to DNA damage responses, cell growth, cell differentiation, and cell death were upregulated in high-dose-rate–irradiated Lgr5+ cells. Interestingly, pathways related to apical junctions and extracellular signaling were upregulated in low-dose-rate–irradiated Lgr5+ cells (Otsuka et al., manuscript in preparation). Biological events involving apical junctions are known to play an important role in the extrusion of transformed cells surrounded by normal epithelial cells through a process called ‘cell competition’ [33, 34]. We speculated that cell competition through apical junctions and extracellular ligands might contribute to dose-rate effects in Lgr5+ cell replenishment. Based on our findings, we propose a possible mechanism behind the dose-rate effect on colonic stem cells (Fig. 2). To understand the molecular mechanism underlying the dose-rate effect, we hypothesized that genes specifically activated in low-dose-rate–irradiated Lgr5+ stem cells determine the consequence of the dose-rate effect. We created a list of genes that are significantly upregulated in low-dose-rate–irradiated colonic Lgr5+ stem cells compared with non-irradiated and high-dose-rate–irradiated colonic Lgr5+ stem cells. We found 69 genes that were significantly upregulated in low-dose-rate–irradiated colonic Lgr5+ stem cells, which we named DREDGEs (Dose-Rate Effect Determining GEnes). In future studies, we aim to identify the features of DREDGEs to uncover their functions in the dose-rate effect on colonic Lgr5+ stem cell replenishment.

Fig. 2.

A possible model of dose-rate effect in the replenishment of Lgr5+ stem cells. After high-dose-rate irradiation, the cell cycle of colonic Lgr5+ stem cells immediately stalls due to DNA damage, and some cells die or differentiate. As the number of colonic Lgr5+ stem cells decrease after irradiation, the Lgr5+ stem cell pool is replenished by the surviving Lgr5+ stem cells and/or radioresistant, quiescent stem cells. This unscheduled growth stimulation may result in the accumulation of mutations. After low-dose-rate irradiation, responses related to cell competition are induced in the colonic Lgr5+ stem cell pool, and some cells may be excluded from the pool to maintain the integrity of tissue stem cells.

CONCLUSION

Although radiation-specific mutation spectra have been published previously [35-37], these reports were based on high-dose-rate radiation. Recent studies have demonstrated that dose-rate-specific mutation spectra are important for studying cancer risk during exposure to various dose-rate irradiations [38]. Although the importance of the dose-rate effect on tissue stem cells is considered in radiation protection [39], the mechanism behind the dose-rate effect is still unknown. Our approach for investigating this mechanism by studying the features of DREDGEs may help us understand how tissue stem cells maintain their integrity during exposure to low-dose-rate radiation.