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Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

ABSTRACT: Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.

SUBMITTER: Alessio N

PROVIDER: S-EPMC5323029 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

Alessio Nicola N Capasso Stefania S Di Bernardo Giovanni G Cappabianca Salvatore S Casale Fiorina F Calarco Anna A Cipollaro Marilena M Peluso Gianfranco G Galderisi Umberto U

Cell cycle (Georgetown, Tex.) 20160428 3

Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed p ...[more]

PMID: 27124644

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Project description:ObjectivesOne of the major side effects of radiotherapy for treatments of the head and neck cancer is the radiation-induced dysfunction of salivary glands. The aim of the present study is to investigate the efficacy of deferoxamine (DFO) to restore the secretory function of radiation-damaged salivary glands in mice.MethodsDFO (50 mg/kg/d) was administered intraperitoneally in C57BL/6 mice for 3 days before and/or after point-fixed irradiation (18 Gy) of submandibular glands. The total 55 mice were randomly divided into: (1) Normal group: mice received no treatment (n = 5); (2) Irradiation group (IR): mice only received irradiation (n = 5); (3) Pre-DFO group (D+IR) (n = 10); (4) Pre+Post DFO group (D+IR+D) (n = 10); (5) Post-DFO group (IR+D) (n = 10); (6) For each DFO-treated group, the mice were intraperitoneally injected with 0.1 ml sterilized water alone (by which DFO was dissolved) for 3 days before and/or after irradiation, and served as control. Sham1: Pre-sterilized water group (n = 5); sham2: Pre+Post sterilized water group (n = 5); sham3: Post-sterilized water group (n = 5). The salivary flow rate (SFR) was assessed at 30th, 60th and 90th day after irradiation, respectively. After 90 days, all mice were sacrificed and their submandibular glands were removed for further examinations.ResultsThe salivary glands showed remarkable dysfunction and tissue damage after irradiation. DFO restored SFR in the irradiated glands to a level comparable to that in normal glands and angiogenesis in damaged tissue was greatly increased. DFO also increased the expression levels of HIF-1α and VEGF while reduced apoptotic cells. Furthermore, Sca-1+cells were preserved in the salivary glands treated with DFO before IR.ConclusionsOur results indicate DFO could prevent the radiation-induced dysfunction of salivary glands in mice. The mechanism of this protective effect may involve increased angiogenesis, reduced apoptosis of acinar cells and more preserved stem cells.

Project description:Radiation-induced late side effects such as cognitive decline and normal tissue complications can severely affect quality of life and outcome in long-term survivors of brain tumors. Proton therapy offers a favorable depth-dose deposition with the potential to spare tumor-surrounding normal tissue, thus potentially reducing such side effects. In this study, we describe a preclinical model to reveal underlying biological mechanisms caused by precise high-dose proton irradiation of a brain subvolume. We studied the dose- and time-dependent radiation response of mouse brain tissue, using a high-precision image-guided proton irradiation setup for small animals established at the University Proton Therapy Dresden (UPTD). The right hippocampal area of ten C57BL/6 and ten C3H/He mice was irradiated. Both strains contained four groups (nirradiated = 3, ncontrol = 1) treated with increasing doses (0 Gy, 45 Gy, 65 Gy or 85 Gy and 0 Gy, 40 Gy, 60 Gy or 80 Gy, respectively). Follow-up examinations were performed for up to six months, including longitudinal monitoring of general health status and regular contrast-enhanced magnetic resonance imaging (MRI) of mouse brains. These findings were related to comprehensive histological analysis. In all mice of the highest dose group, first symptoms of blood-brain barrier (BBB) damage appeared one week after irradiation, while a dose-dependent delay in onset was observed for lower doses. MRI contrast agent leakage occurred in the irradiated brain areas and was progressive in the higher dose groups. Mouse health status and survival corresponded to the extent of contrast agent leakage. Histological analysis revealed tissue changes such as vessel abnormalities, gliosis, and granule cell dispersion, which also partly affected the non-irradiated contralateral hippocampus in the higher dose groups. All observed effects depended strongly on the prescribed radiation dose and the outcome, i.e. survival, image changes, and tissue alterations, were very consistent within an experimental dose cohort. The derived dose-response model will determine endpoint-specific dose levels for future experiments and may support generating clinical hypotheses on brain toxicity after proton therapy.

Project description:BACKGROUND: We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process. METHODS: We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP. RESULTS: We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger. CONCLUSION: Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

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Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

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Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

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