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Keywords:

  • Irradiation;
  • DNA repair;
  • γ-H2AX;
  • Mesenchymal stem cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We have recently shown that the in vitro differentiation of human mesenchymal stem cells (hMSCs) was accompanied by an increased sensitivity toward apoptosis; however, the mechanism responsible for this shift is not known. Here, we show that the repair of DNA double-strand breaks (DSBs) was more rapid in undifferentiated hMSCs than in differentiated osteoblasts by quantification of the disappearance of γ-H2AX foci in the nuclei after γ-irradiation-induced DNA damage. In addition, there was a marked and prolonged increase in the level of nuclear Ku70 and an increased phosphorylation of DNA-PKcs. This was accompanied by an augmentation in the phosphorylation of ATM in hMSCs post-irradiation suggesting the nonhomologous end joining repair mechanism. However, when hMSCs were induced to differentiate along the osteogenic or adipogenic pathways; irradiation of these cells caused an expeditious and robust cell death, which was primarily apoptotic. This was in sharp contrast to undifferentiated hMSCs, which were highly resistant to irradiation and/or temozolomide-induced DSBs. In addition, we observed a 95% recovery from DSB in these cells. Our results suggest that apoptosis and DNA repair are major safeguard mechanisms in the control of hMSCs differentiation after DNA damage. STEM CELLS 2013;31:800–807


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

DNA repair is essential for cell survival and function [1]. Recent studies have indicated that stem cells respond to DNA damage by increasing DNA repair activity [2]. This property might favor long-term survival but also results in an accumulation of mutations in stem cells and as such might promote tumorigenesis [3]. Human bone marrow-derived mesenchymal stem cells (hMSCs) are long-lived cells that can differentiate into mesenchymal cells (e.g., adipocytes, chondrocytes, osteoblasts, and hepatocytes) as well as transdifferentiate in vitro into other cell types such as neural cells [4]. Recently, hMSCs have received much attention because of their potential use as therapeutic agents for either regeneration of wound tissue or targeting diseases such as cancer [5]. Human MSCs constitute a niche in the stromal compartment of the bone marrow where these cells play an essential role in the control of the function of the hematopoietic stem cells (HSCs) [6]. It has been suggested that hMSCs could also participate to the tumoral microenvironment in several types of cancers [5, 6].

Most cancer patients receive irradiation and chemotherapy as standard treatments in order to eliminate dividing malignant cells through DNA damage. However, these treatments also affect normal tissue, in particular the bone marrow, inducing severe side effects following irradiation [7]. It is not well understood how hMSCs in either the bone marrow or the microenvironment of a tumor will be affected by cancer therapies [8]. Contradictory results have been published on the survival of hMSCs after irradiation or chemotherapy but it is usually assumed that hMSCs are resistant to γ-irradiation even at high doses [9, 10]. In fact studies have shown that hMSCs from patients, exposed to whole body irradiation followed by allogenic bone marrow transplantation, expressed a complete host profile suggesting that the hMSCs in their niches were most probably radio-resistant [11, 12]. In addition, it has been postulated that hMSCs could protect the patient against irradiation-induced hematopoietic failure [13]. A better understanding of the response of hMSCs to DNA damage could provide new insights into the effects of cancer treatments as well as into the development of treatment-associated side effects. This question is also important in the understanding of how the DNA damage, which accumulates with age leading to either cell death or gene silencing that could affect the capacity of hMSCs to regenerate tissues after injury [14]. Indeed, unrepaired DNA double-stranded breaks (DSBs) result in toxic lesions, chromosomal aberrations, and genomic instability that could give rise to cancers [15]. We have recently shown that hMSCs are resistant to apoptosis when undifferentiated and became sensitive to cell death during the early steps of differentiation [16]. This could be related to the fact that caspases are implicated both in apoptosis and in cell differentiation [17]. Understanding the conditions, under which hMSCs acquire a sensitivity toward cell death is also crucial, both in the development of hMSCs as a therapeutic tool and in the advancement of our understanding of the role of tumor stroma during cancer progression [18].

In this study, we have investigated the induced DNA damage response (DDR) in hMSCs to ionizing irradiation at a dose used in the treatment of gliomas. Cell death, proliferation, and differentiation were analyzed either in undifferentiated hMSCs or during osteogenic differentiation of these cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Materials

Unless stated otherwise, all cell culture material was obtained from Gibco (Life Technologies, Cergy Pontoise, France) and chemicals were from Sigma-Aldrich (St. Louis, MO). The bone marrow samples used in this study were obtained from healthy donors operated at the Department of Orthopedics at “Centre Hospitalier Universitaire de Nantes France.” The average age of patients was 41 ± 3 years (ranging from 25 to 56 years; six males and eight females). All human samples were obtained according to the recommendations of the French national ethics committee.

Methods

Human MSC Isolation and Cell Culture

The bone marrow cells were isolated by density gradient centrifugation (Ficoll). The cells collected at the interface were cultured in α-MEM containing with ribonucleosides and deoxyribonucleosides supplemented with 20% fetal calf serum, with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (complete hMSC medium) in an atmosphere of 5% CO2 and 95% humidity. Human MSC cultures were used between passage 2 and 10 passage. Cultures were kept at subconfluent levels (approximately 75% confluence) and passaged every 5–7 days. The glioblastoma U251 cell line was cultured in Dulbecco's modified Eagle's medium (4.5 g/l glucose) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 and 95% humidity. γ-Irradiation was carried out in a Faxitron CP160 irradiator (Faxitron X-ray Corporation) at a dose rate of 1.48 Gy/minute. Human fibroblasts were obtained and cultured as described early [16, 19]. Cells were cultured under hypoxia (1% O2, 5% CO2, and 37°C) using a Sci-tive Hypoxia Workstation (Awel International, Blain, France).

Differentiation of hMSCs

Human MSCs differentiation into osteoblasts was induced in vitro by culturing the cells in NH OsteoDiff medium (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 21 days. Osteogenic differentiation was detected by staining with alizarin red, which binds to mineralized bone. For adipogenic differentiation, the cells were cultured for 3 weeks in hMSC Adipogenic Bullet kit (Lonza, Slough, U.K.). Adipogenic differentiation was detected by staining the cells with oil red O.

Western Blotting

Total proteins were extracted in 1% Nonidet P40, 0.5% sodium-deoxycholate, and 0.1% SDS supplemented with protease inhibitor cocktail from Roche Diagnostics (Mannheim, Germany). The protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Protein extracts were separated on SDS-PAGE, transferred onto poly vinylidene fluoride membrane (Millipore, St. Quentin-Yvelines, France), and revealed by Immobilon Western chemiluminescent horse radish peroxydase substrate (Millipore). Primary antibodies were used at 1/1,000 dilution: mouse monoclonal anti-actin (clone C4, MAB1501, Millipore), mouse monoclonal anti-phospho-ATMSer1981 (DR1002, Millipore), rabbit polyclonal anti-phospho-DNA-PKcsSer2056 (ab18192 Abcam, Cambridge, U.K.), mouse monoclonal anti-Ku70 (MS-329-P1, Thermo Scientific, France), and mouse monoclonal anti-Ku80 (MS-285-P1, Thermo Scientific). HRP-conjugated secondary antibodies were from Bio-Rad. Quantification was performed with the software ImageJ.

Immunocytochemistry

Cells were grown on gelatin-coated glass coverslips. Cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% SDS for 10 minutes, blocked with 3% bovine serum albumin for 20 minutes, and then incubated with primary antibodies for 1 hour followed by secondary Alexa antibodies (Molecular Probes-Life Technologies) for 1 hour. Cells were finally mounted with Mowiol polymerizing solution and observed under a confocal microscope (Leica TCS-SP1). Primary mouse monoclonal anti-H2AXSer139 was obtained from Millipore (05-636). The number of γ-H2AX foci was determined using ImageJ and the three-dimensional object counter plugin. Briefly, the acquisition of images was done using a z-stack of 0.25 μm per image. The stack was opened and threshold determined so that each foci was equal to or more than 15 × 15 pixels (1 μm2).

Senescence

Senescence was assessed by x-gal staining using the senescence cell histochemical staining kit (Sigma-Aldrich) according to the manufacturer's instructions.

FACScan Analysis

The phenotype of hMSCs was monitored by flow cytometry. For phenotypic analysis, conjugated and nonconjugated antibodies were used (cf. supporting information Table S1). For nonconjugated antibodies, the corresponding secondary antibody was used. Briefly, 2 × 105 cells were resuspended in complete medium for 30 minutes at 4°C. For intracellular staining, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized in phosphate buffered saline containing 0.5% saponin. The cells were incubated with the primary antibody for 30 minutes at 4°C in phosphate buffered saline, 0.25% saponin and then, where necessary, the secondary antibody was added for 30 minutes at 4°C. Cells were washed twice in phosphate buffered saline before analyzed on a FACScalibur (BD Biosciences, Le Pont de Claix, France) using Cell Quest Pro software. The appropriate isotype controls were included and a minimum of 10,000 events were acquired for each condition. The debris was excluded from the analysis according to their forward scatter count/side scatter count properties. BD ApoAlert APO 2.7-phycoerythrin (BD Biosciences) was used to determine the percentage of apoptotic cells according to the manufacturer's instructions.

For cell cycle analyses, the cells were pulsed with BrdU for 20 minutes at 37°C. After ethanol fixation and acidic DNA denaturation, cells were stained with an anti-BrdU FITC antibody (51-33284X, BD Biosciences) and propidium iodide and then analyzed by flow cytometry.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Acquisition of Sensitivity to Irradiation During the Differentiation of hMSCs In Vitro

Human MSCs were isolated from healthy human donors as previously described [16]. Cells were isolated from human bone marrow aspirates and cultured as described by Pittenger et al. [4]. After 2 weeks of culture, the cell population was characterized by flow cytometric analysis for the expression of CD105, CD90, and CD44 markers and the absence of markers of the hematopoietic lineage, including the lipopolysaccharide receptor CD14, CD34, and the leukocyte common antigen CD45. Flow cytometry parameters used in this study are listed in supporting information Table S1. Osteogenesis was induced and visualized as described in Materials and Methods. Of note, after 1 week in the osteogenic medium, no morphological signs of differentiation were visible as yet (Fig. 1A) and complete differentiation was observed only after 3 weeks of culture (Fig. 1B). Human MSCs cultured in differentiation medium for 1 week will be further referred to as predifferentiated (prediff.) hMSCs and after 3 weeks of culture as differentiated (diff.) hMSCs.

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Figure 1. Influence of osteogenic differentiation medium on hMSCs morphology and apoptosis. (A): Morphological aspects of hMSCs cultured for 1 week in complete media or in osteogenic differentiation medium (prediff.). (B): Osteogenic differentiation was determined by alizarin red (binds to mineralized bone) in hMSC cultures after 3 weeks of incubation in either complete media or in osteogenic differentiation medium (diff.). (C): Human MSCs cultured in complete hMSC media or in osteogenic differentiation medium for 1 week (prediff.) were subjected or not to 5 Gy γ-irradiation and then cell viability was determined by trypan blue exclusion at the indicated times. The results are the mean ± SD of three independent experiments. Abbreviation: hMSCs, human mesenchymal stem cells.

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Undifferentiated and predifferentiated hMSCs were irradiated with a single dose of 5 Gy as described in Materials and Methods. This dose has been shown to efficiently trigger apoptosis in hematopoietic progenitor cells [20]. Quantification of cell death showed that predifferentiated hMSCs were sensitive to apoptosis as compared to undifferentiated hMSCs, which were resistant to irradiation-induced cell death (Fig. 1C). Note that differentiated hMSCs were sensitive to cell death inducers such as staurosporine or etoposide as previously described [6] as well as γ-irradiation.

To verify whether this effect was related to the type of cellular differentiation, we repeated this experiment with hMSCs differentiated along the adipogenic pathway (Fig. 2A). As illustrated in Figure 2B, hMSCs predifferentiated into adipocytes were sensitive to γ-irradiation. To verify whether factors present in the differentiation culture medium were capable of protecting the cells against radiation, hMSCs predifferentiated into either osteoblasts or adipocytes for 1 week were cultured in complete hMSCs medium and then γ-irradiation. We did not find any difference in the number of cell death observed over the first 48 hours (data not shown) ruling out a possible effect of the different media in radio-resistance. However, as shown in Figure 2C, the surviving predifferentiated hMSCs appeared to be more senescent when cultured in complete hMSC media.

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Figure 2. Influence of adipogenic differentiating medium on hMSCs morphology and apoptosis. (A): Morphological aspects of human MSCs (hMSCs) cultured for 3 weeks in complete hMSC media or in adipogenic differentiation medium. Adipogenic differentiation was determined after 3 weeks of incubation in either complete hMSC media or in adipogenic differentiation medium using oil red O. (B): Human MSCs cultured in complete media or in adipogenic differentiation medium for 1 week (prediff.) were subjected or not to 5 Gy γ-irradiation and then cell viability was determined by trypan blue exclusion at the indicated times. The results are the mean ± SD of three independent experiments. (C): Human MSCs were cultured in osteogenic or adipogenic differentiation media for 1 week and then cultured for another week in complete hMSC medium before being subjected to 5 Gy γ-irradiation. One week later, cellular senescence was determined by x-gal staining. (D): Human fibroblasts cultured in complete hMSC media were subjected or not to 5 Gy γ-irradiation and then cell viability was determined by trypan blue exclusion at the indicated times. The results are the mean ± SD of three independent experiments. Abbreviations: IR, irradiation; MSCs, mesenchymal stem cells.

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To evaluate a possible “niche” effect, we analyzed the effect of hypoxia on the response of hMSCs to γ-irradiation. We found that hypoxia (i.e., 1% O2) provided a limited effect on hMSCs survival after γ-irradiation (supporting information Fig. S1). Finally, we asked if the use of primary cultures was important in the response to γ-irradiation, we study the response of human fibroblasts cultured in hMSCs culture medium. As illustrated in Figure 2D, unlike hMSCs, human fibroblasts were sensitive to γ-irradiation even in the presence of complete hMSC medium. Altogether, these results suggest that the differentiation related to γ-irradiation sensitivity was specific to hMSCs.

Differential Induction of γ-H2AX Foci in Undifferentiated and Predifferentiated hMSCs Upon Irradiation

The response to DNA damage resulting in DSBs involves an orchestrated assembly of proteins that has been used to develop DSBs detection assays [21, 22]. Once DNA damage is detected, there is activation of the DDR, the components of which can be divided into four functional groups: the DNA damage sensors, the signal transducers, the repair effectors, and the cell arrest or death effectors.

γ-H2AX foci analyses take advantage of an early event in DDR, namely the phosphorylation of the histone H2AX [21, 22]. This assay is highly sensitive and can potentially detect all induced DSBs [22] and as such is generally used as a surrogate marker of DSBs. Immunofluorescence was used to visualize and quantify DDR kinetics using γ-H2AX foci in two different models: undifferentiated hMSCs and the human glioma cell line U251. As shown in Figure 3, γ-H2AX foci increased after irradiation in both cultures but the amount of γ-H2AX foci decreased in hMSCs (Fig. 3A) after 12 hours while the number in the human glioma cell line U251 (Fig. 3B) remained constant over time (Fig. 3C). Contrary to hMSCs, cell death was induced in U251 cells 12 hours post-irradiation, suggesting a correlation between the failure of DNA repair and the induction of cell death (data not shown) [23].

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Figure 3. Difference in DNA repair activity between hMSCs and U251. hMSCs (A) and human glioma cell line U251 (B) were subjected to γ-irradiation (5 Gy) and then at different time points the cells were fixed and labeled with a monoclonal mouse anti-H2AXser139 and the nuclei were stained with Dapi. (C): The number of γ-H2AX foci in the nuclei was quantified using ImageJ and the three-dimensional object counter plugin. An average of 100 nuclei was analyzed at each time point. Abbreviation: hMSCs, human mesenchymal stem cells.

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Cells repair DNA damage after irradiation primarily by two mechanisms: nonhomologous end joining (NHEJ) repair or homologous recombination (HR) repair. We thus determined in hMSCs the mechanism of repair of DSB. Since two protein complexes made up of Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/Ligase IV/XLF effectuate NHEJ repair of DSB, we analyzed the expression of Ku80, Ku70, and DNA-PKcs in hMSCs after γ-irradiation. As shown in Figure 4 the expression of Ku70 increased in hMSCs after irradiation while that of Ku80 remained constant. The increase in the expression of Ku70 was visible at 30 minutes and peaked at 2 hours and remained at this level until 24 hours. Note that the expression of phospho-DNA-PKcsSer2056 increased after 30 minutes and then returned to basal levels after 2 hours. Furthermore, the level of phospho-ATMSer1981 followed the same pattern of expression of that of phospho-DNA-PKcsSer2056.

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Figure 4. DNA repair activity in undifferentiated hMSCs. hMSCs were irradiated (5 Gy) and then at the different times indicated cell lysates were prepared and separated in SDS-PAGE to determine the expression of pDNA-PKcsSer2056, Ku80, Ku70, and pATMSer1981. Abbreviation: hMSCs, human mesenchymal stem cells.

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Next, we asked if predifferentiated hMSCs responded in a similar way to irradiation as undifferentiated hMSCs. As shown in Figure 5A, a 5 Gy γ-irradiation induced an accumulation of γ-H2AX foci with a similar intensity and kinetics between undifferentiated and predifferentiated hMSCs since in both cases, a peak was observed after 1 hour. However, the number of foci decreased more expeditiously in undifferentiated hMSCs than in predifferentiated cells: after 24 hours, the amount of γ-H2AX foci remained high in the surviving predifferentiated hMSCs (Fig. 5B) and returned to normal only after 72 hours in the surviving cells (data not shown) while in undifferentiated hMSCs, no foci could be observed after 24 hours (Fig. 5A).

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Figure 5. DNA repair activity in undifferentiated and differentiated hMSCs. (A): Human MSCs were cultured in either complete media or in osteogenic differentiation media for 3 weeks and then subjected to 5 Gy γ-irradiation. At different time points, the cells were fixed and labeled with a monoclonal mouse anti-H2AXSer139 and the nuclei were stained with Dapi. (B): The number of γ-H2AX foci in the nuclei was quantified using ImageJ and three-dimensional Object Counter plugin. An average of 100 nuclei was analyzed at each time point. The data presented are the mean ± SD. (C): To determine the effect of γ-irradiation on osteogenic differentiation, hMSCs cultured in complete medium were subjected to γ-irradiation (5 Gy) and then cultured in osteogenic differentiation medium (hMSCs + 5 Gy + diff.). As a control, hMSCs were either cultured in complete media (hMSCs), in osteogenic differentiation media (hMSCs + diff.), or hMSCs cultured in complete media were irradiated (hMSCs + 5 Gy). The number of cells in the different cultures was determined at the indicated times. The data presented are duplicate values of three independent experiments. (D): To determine the effect of irradiation on the cell cycle, hMSCs were subjected or not to γ-irradiation (5 Gy) and 24 hours later the cells were stained with BrdU and propidium iodide. Cells were analyzed by flow cytometry and the number of cells in G1, S, and G2/M was quantified p < 0.0001. Abbreviation: hMSCs, human mesenchymal stem cells.

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Recent results have suggested a link between DNA repair and differentiation, in particular on the unanticipated roles of DDR in the regulation of precursor or stem cell differentiation [24]. We thus examined the effect of irradiation on the osteogenic differentiation of hMSCs. As shown in Figure 5C, irradiated hMSCs were cultured for 6 days in differentiation medium. Within 1 week, these irradiated hMSCs underwent massive and rapid cell death even in absence of additional apoptotic inducers. These results suggest that hMSCs cannot sustain survival upon differentiation after irradiation and that acquisition of sensitivity to apoptosis is concomitant of early differentiation. As depicted in Figure 5D, analyses of the cell cycle showed that 24 hours after irradiation there were significantly less cells in phase S (con vs. irradiated: 9 vs. 2.9; p > .0005) and more cells in G2/M (con vs. irradiated: 7.6 vs. 10.9; p = .007).

DDR Response of hMSCs and U251 Cells to Temozolomide and γ-Irradiation

Several studies have shown that hMSCs can survive irradiation but little is known about its survival upon the combination of chemical- and radiation-induced DNA damage, a classic therapeutic regimen in the treatment of brain tumors [25]. This question is significant since hMSCs are precursors of the stroma associated with many cancers, and as such could modulate the immune system and produce tumor-associated myofibroblasts [26]. We analyzed the response of hMSCs to another DNA damaging agent, temozolomide (TMZ) an oral alkylating agent, which is used in the treatment of Grade IV astrocytoma, the most aggressive brain tumor in adult [25]. TMZ rapidly methylates DNA, causing DNA damage whereby triggering cell death [25].

First, we analyzed the effect of TMZ and/or γ-irradiation on the viability of U251 glioma cells and on the formation of DSBs visualized by the formation of γ-H2AX foci (Fig. 6). TMZ at 25 μM, a concentration close to the therapeutic dose, was added to U251 cells and as shown in Figure 6A triggered the formation of γ-H2AX foci, which increased over time after 8 hours until 48 hours where cell death became dominant. Similar results were obtained when U251 cells were subjected to 5 Gy irradiation; however, treatment of a combination of irradiation and TMZ resulted in a significant increase (p = .0019) in cell death (Fig. 6B) as compared to irradiation alone or TMZ alone, as previously described [17]. Next, we analyzed the effect of these treatments to DNA repair and cell death induction in hMSCs cultures (Fig. 6). A treatment of hMSCs with 25 μM TMZ induced a limited number of γ-H2AX foci that persisted after 6 hours. However, when hMSCs were treated with 25 μM TMZ prior to γ-irradiation, the induction of γ-H2AX foci was less important than that induced by irradiation alone (Fig. 7A). However, in all cases, no difference in the induction of cell death was observed between the different treatments suggesting that apoptosis was efficiently blocked in these cells (Fig. 7B). These results suggest that the combination of treatment (i.e., TMZ and γ-irradiation), which is usually used in the treatment of gliomas, does not affect the survival of hMSCs.

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Figure 6. Comparison of DNA repair and sensitivity toward cell death in U251 cells. (A): The human glioma U251 cells were treated with 25 μM TMZ and then cells were fixed and labeled with the anti-γ-H2AX at the indicated times and (B) quantification of the γ-H2AX foci was done as described in Figure 3. An average of 80 cells were counted per time point. (C): U251 cells were both cultured in the absence or presence of 25 μM TMZ or γ-irradiated (5 Gy) and then cultured in the presence of 25 μM TMZ. Cell viability was assessed by trypan blue exclusion. Data are presented as mean ± SD of duplicate values of three independent experiments. **, Statistical analyses of the percentage of cell death between irradiated cells and a treatment TMZ + irradiation of p = .0019. Abbreviations: IR, irradiation; TMZ, temozolomide.

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Figure 7. Comparison of DNA repair with cell death induction in human mesenchymal stem cells (hMSCs) by TMZ and/or irradiation. (A): Undifferentiated hMSCs were both cultured in the presence of 25 μM TMZ or irradiated (5 Gy) and then cultured in the presence of TMZ (25 μM). The nuclei were labeled with an anti-H2AXSer139 to quantify the number of γ-H2AX foci as described above. (B): Simultaneously, the percentage of cell death under the different conditions of (A) was determined by trypan blue exclusion as described in previous figures. Abbreviations: IR, irradiation; TMZ, temozolomide.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

It has been postulated that aging and cancer are the consequences of unrepaired DNA damage due mainly to the accumulation of reactive oxygen species from cellular metabolism and ultraviolet light from the sun [27]. Nuclear DNA damage might contribute to aging by increasing cell death, cellular senescence, or more generally by decreasing cell functions [25]. When cells avoid the latter processes and accumulate DNA mutations, they are at greater risk to undergo oncogenic transformation [28]. In proliferating cells, DNA damage can also be limited through several mechanisms that ensure DNA replication fidelity [29]. In addition, another major concern in cancer and cancer therapies is genomic instability and recently this problem has also become an obstacle in regenerative medicine therapies. It has been shown that hMSCs propagated in culture develop aneuploidy and other DNA rearrangements over time that renders these cells unsuitable for clinical use [30]. Thus, it is important to understand the mechanisms required to maintain genomic stability. The source of DNA damage and the type of breaks produced would determine the kind of DDR activated. DNA damage may be identified by changes in the chromatin structure after DSBs. DDR components would be recruited to the sites of DNA damage after the formation of foci. In the case of DSBs, the ATM-ATR-DNA PK pathway induces the phosphorylation of the histone variant γ-H2AX on the chromatin at DSB sites. This results in ubiquitin-mediated recruitment of DSB repair factors and additional chromatin modifying factors, which act to amplify the DNA damage signal, recruiting even more repair-associated proteins [31, 32]. The form of DSB repair with the highest fidelity is HR repair, which uses homologous sequences from a sister chromosome as a template for repairing damaged DNA. NHEJ represents a more error-prone form of DNA repair with faster repair kinetics than HR repair. A recent study by the group of Valerie [33] showed that neural progenitors used the NHEJ DNA repair and that the rate of NHEJ progressively increased while the fidelity of repair decreased during the differentiation into astrocytes.

The latter processes are obviously less available in slow or nondividing cells, such as stem cells. Thus, adult stem cells are thought to accumulate DNA damage during their quiescent or resting state in vivo [2, 28]. However, there is a certain heterogeneity of the apoptotic response to DNA damage among of stem cells as some die rapidly after irradiation while others are resistant even to high intensity radiations [28]. We have recently shown that adult hMSCs are highly resistant to apoptosis unless they are engaged into differentiation [16]. Recent results have shown that HSCs are radio-protected by different mechanisms during quiescent and proliferating stages. However, in quiescent HSCs, DNA damage is associated with genomic rearrangements and as such HSCs are more susceptible to error-prone DNA repair and mutagenesis [3].

In this study, we have shown that adult hMSCs isolated from bone marrow are resistant to radiation unless differentiation is induced (Figs. 1, 2). We found that in undifferentiated hMSCs possess a high DNA double-strand break (DSB) repair activity as viewed by γ-H2AX foci (Fig. 3). When engaged into differentiation, hMSCs loose this capacity and DNA breaks are followed by cell death (Fig. 5). We also analyzed the response of hMSCs to another type of DNA damage, for example, that induced by TMZ, and as shown in Figure 6, similar results were found. This result suggests that hMSCs possess an intrinsic resistance to DNA damage. Of note, irradiated hMSCs undergo apoptosis when differentiation is induced (Fig. 5). Taken together, our data suggest that irradiation-induced DNA damage is readily repaired in hMSCs but impaired their differentiation through the induction of apoptosis. NHEJ can occur at any point in the cell cycle. This mechanism of repair is initiated by recognition and processing of the DSBs by the Ku80/Ku70/DNA-PKcs/Artemis heterodimer. This activates DNA-PKcs, which mediates recruitment of end processing enzymes and polymerase. DNA ligase IV mediates religation of the broken ends of the DNA. This method of repair is error prone and results in sequence deletion and mutations. As appose to HR repair (active in the U251 cells), which uses the sister chromatid as the template for repair and therefore can only occur during S-phase or G2. HR is initiated by the MRE11-RAD50-NBS1 complex, which binds to and processes the ends of the damaged DNA, generating single strand DNA (ssDNA). The ssDNA invades the template and repair is mediated by polymerase, nuclease, helicase, and ligase activity, followed by resolution of the structure. This process can be considered as a safeguard against unwanted mutagenesis and that inhibition of apoptosis in hMSCs will enhance the risk of oncogenesis may be more than DNA damage itself. In addition, our results show that the regimen used for the treatment of gliomas, which involves both radiation- and chemo-induced DNA damages, has no affect on hMSCs survival. Human MSCs possess ambivalent roles toward tumoral growth because of their capacity to participate in the non-neoplastic compartment, their immuno-modulatory properties and/or secretory activity [17].

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Based on our results, one can postulate that irradiated hMSCs could still be used as therapeutic vehicle but would not be able to differentiate in the tumoral microenvironment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank Emeline Brocard for technical assistance and Philippe Hulin from the “Cellular and Tissular Imaging Core Facility (MicroPICell), Université de Nantes” for his aid with all the microscopic analyses. This study was supported by a special program from “Equipe Labelisée la Ligue Contre le Cancer.”

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0541_sm_SupplFigure1.pdf41KFigure S1: Effect of hypoxia on hMSCs survival after γ-irradiation. Human MSCs were cultured under normoxia (20% O2) or hypoxia (1% O2) for 48 h before γ- irradiation. Cells were counted at the indicated times. The results are the mean ± SD of a representative experiment repeated three times.
sc-12-0541_sm_SupplTable1.pdf37KTable S1: Antibodies used to characterize hMSCs as a percentage of positive cells.

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