Séverine Cruet-Hennequart

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.

Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase η-deficient human cells treated with cisplatin and oxaliplatin

The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin. Poleta-deficient cells are more sensitive to both drugs than are normal cells. In poleta-deficient cells, drug treatment leads to prolonged S-phase arrest, and increased phosphorylation of the phosphatidylinositol-3-kinase-related protein kinase (PIKK) substrates Chk1, p95/Nbs1 and RPA2, the 34kDa subunit of replication protein A. Cisplatin- and oxaliplatin-induced hyperphosphorylation of RPA2, and association of the hyperphosphorylated protein with chromatin, is elevated in poleta-deficient cells. Cisplatin-induced phosphorylation of RPA2 on serine 4/serine 8, but not on serine 33, is inhibited by the DNA-PK inhibitor, NU7441, but not by the ATM inhibitor, KU-55933. Cisplatin-induced DNA-PK-dependent hyperphosphorylation of RPA2 on serine 4/serine 8 occurs after recruitment of RPA to chromatin, as determined by immunofluorescence and by subcellular fractionation. ATR is required both for recruitment of RPA2 to chromatin and its subsequent hyperphosphorylation on serine 4/serine 8 by DNA-PK, since CGK733, an inhibitor of ATM and ATR, blocked both recruitment and hyperphosphorylation. Thus, increased sensitivity to cisplatin and oxaliplatin in DNA poleta-deficient cells is associated with prolonged S-phase arrest, and enhanced PIKK-signalling, in particular activation of DNA-PK-dependent hyperphosphorylation of RPA2 on serines 4 and 8.

Characterization of the effects of cisplatin and carboplatin on cell cycle progression and DNA damage response activation in DNA polymerase eta-deficient human cells.

Translesion synthesis by DNA polymerase eta (polη) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking polη are sensitive to these agents. To help define the consequences of polη-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking polη. We show that both cisplatin and carboplatin induce strong S-phase arrest in polη-deficient XP30RO cells, associated with reduced expression of cyclin E and cyclin B. PIK kinase-mediated phosphorylation of Chk1, H2AX and RPA2 was strongly activated by both cisplatin and carboplatin, but phosphorylation of these proteins was induced earlier by cisplatin than by an equitoxic dose of carboplatin. Compared to Chk1 and H2AX phosphorylation, RPA2 hyperphosphorylation on serine4/serine8 is a late event in response to platinum-induced DNA damage. We directly demonstrate, using dual-labeling flow cytometry, that damage-induced phosphorylation of RPA2 on serine4/serine8 occurs primarily in the S and G2 phases of the cell cycle, and show that the timing of RPA2 phosphorylation can be modulated by inhibition of the checkpoint kinase Chk1. Furthermore, Chk1 inhibition sensitizes polη-deficient cells to the cytotoxic effects of carboplatin. Both hyperphosphorylated RPA2 and the homologous recombination protein Rad51 are present in nuclear foci after cisplatin treatment, but these are separable events in individual cells. These results provide insight into the relationship between cell cycle regulation and processing of platinum-induced DNA damage in human cells when polη-mediated TLS is compromised.

DNA Polymerase η, a Key Protein in Translesion Synthesis in Human Cells

Genomic DNA is constantly damaged by exposure to exogenous and endogenous agents. Bulky adducts such as UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA present a barrier to DNA synthesis by the major eukaryotic replicative polymerases including DNA polymerase delta. Translesion synthesis (TLS) carried out by specialized DNA polymerases is an evolutionarily conserved mechanism of DNA damage tolerance. The Y family of DNA polymerases, including DNA polymerase eta (Pol eta), the subject of this chapter, play a key role in TLS. Mutations in the human POLH gene encoding Pol eta underlie the genetic disease xeroderma pigmentosum variant (XPV), characterized by sun sensitivity, elevated incidence of skin cancer, and at the cellular level, by delayed replication and hypermutability after UV-irradiation. Pol eta is a low fidelity enzyme when copying undamaged DNA, but can carry out error-free TLS at sites of UV-induced dithymine CPDs. The active site of Pol eta has an open conformation that can accommodate CPDs, as well as cisplatin-induced intrastrand DNA crosslinks. Pol eta is recruited to sites of replication arrest in a tightly regulated process through interaction with PCNA. Pol eta-deficient cells show strong activation of downstream DNA damage responses including ATR signaling, and accumulate strand breaks as a result of replication fork collapse. Thus, Pol eta plays an important role in preventing genome instability after UV- and cisplatin-induced DNA damage. Inhibition of DNA damage tolerance pathways in tumors might also represent an approach to potentiate the effects of DNA damaging agents such as cisplatin.

Radiation-Induced Alterations of Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O2 and 3% O2 conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O2 tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O2 tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.

Human mesenchymal stem cells (hMSCs) as targets of DNA damaging agents in cancer therapy.

Human mesenchymal stem cells (hMSCs) consist of cells that can differentiate into mesenchymal tissues, including osteoblasts, adipocytes and chondrocytes. hMSCs constitute a particular stem cell niche in the stromal compartment of the bone marrow, and also play a role in maintaining the normal function of haematopoietic stem cells. Furthermore, hMSCs localise to solid tumours, and can modulate cancer cell function through secretion of paracrine signals. While hMSCs, either in the bone marrow, or in the microenvironment of a tumour, will be targeted by DNA damaging agents used in cancer therapy, the response of the hMSC population to DNA damage is not well understood. In their role as progenitor cells, genomic DNA damage to hMSCs during cancer therapy could generate a population of surviving cells that can go on to give rise to secondary tumours. 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 secondary cancers. The article will review the relationship of hMSCs to cancer, with a focus on the response of hMSCs to DNA damaging agents.

Impact of Therapeutic Irradiation on Healthy Articular Cartilage

Radiation-induced complications in bone and cartilage are of increasing concern due to potential long-term effects in cancer survivors. Healthy articular cartilage may be exposed to radiation during either chondrosarcoma treatment or in-field radiotherapy of tumors located in close proximity to articulation. Cartilage exposed to radiation undergoes bone differentiation and senescence, which can lead to painful and disabling sequelae that can impair patient quality of life. An understanding of the biological processes involved in healthy cartilage response to radiotherapy may not only optimize the delivery of therapeutic radiation but also reduce the risk of long-term sequelae in irradiated cartilage. Over the last few decades, radiobiology studies have focused primarily on signaling and repair of DNA damage pathways induced by ionizing radiation in immortalized cells under conditions dramatically different from human homeostasis. This research needs to be continued and broadened, since the range of normal tissue responses to radiation exposure is still not fully understood, despite being recognized as the major limiting factor in the rupture of tissue homeostasis after radiotherapy. Human articular cartilage is an avascular tissue with low intracellular oxygen levels and is comprised of a single cell lineage of chondrocytes embedded in a highly dense and structured extracellular matrix. These relatively unique features may impact inherent cell radiation sensitivity and suggests that canonical cell responses to ionizing radiation may not be applicable to articular cartilage. Despite the number of studies in this field, radiation-induced modifications of chondrocyte proteome remain unclear because of the dramatic variability in reported experimental conditions. In this review, we propose to introduce cartilage tissue physiology and microenvironment concepts, and then present a comprehensive synthesis of cartilage radiation biology.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

G2/M Arrest Sensitises Erythroid Leukemia Cells to TRAIL-induced Apoptosis

Erythroidl eukemia is a heterogeneous disease with very poor prognosis. It may arise de novo, secondary to myelodysplastic syndrome, blast crisis phase of chronic myeloid leukemia, or after cytotoxic therapy of acute myeloid leukemia. The current mainstream treatment of erythroleukemia is cytarabine and anthracyclin-based chemotherapy or bone marrow transplantation. In the current study we found that cytarabine or inhibition of the DNA-damage-activated protein kinase, ATM, induce G2/M arrest and sensitised K562 erythro leukemia cells to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Arresting cells in G2/M with microtubule-disrupting drugs also enhanced TRAIL-sensitivity. Synchronisation or separation of the leukemia cells in different stages of the cell cycle by elutriation confirmed that the cells in G1 and G2/M were sensitive to TRAIL. Interestingly, this sensitivity was associated with cell cycle-dependent oscillation of cFLIP expression. In summary, we found that combination of cytostatic drugs with TRAIL can be an effective treatment for erythroid leukemia