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

<p>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 <i>RB1</i> gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors.</p> <p>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 <i>RB1</i> gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated <i>RB1</i> do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated <i>RB1</i> 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 <i>RB1</i>.</p> <p>We extended our analysis to the other members of retinoblastoma gene family: <i>RB2/P130</i> and <i>P107</i>. Also in the MSCs with silenced <i>RB2/P130</i> and <i>P107</i> 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.</p

De-regulated expression of the BRG1 chromatin remodeling factor in bone marrow mesenchymal stromal cells induces senescence associated with the silencing of NANOG and changes in the levels of chromatin proteins

<p>Stem cells have a peculiar chromatin architecture that contributes to their unique properties, including uncommitted status, multi/pluripotency and self-renewal. We analyzed the effect of the de-regulation of the SWI/SNF chromatin remodeling complex in mesenchymal stromal cells (MSC) through the silencing and up-regulation of BRG1, which is the ATPase subunit of the complex. The altered expression of BRG1 promoted the senescence of MSC with suppression of the NANOG transcription, which is part of the transcriptional circuitry governing stem cell functions. To gain insight on the way NANOG was silenced, we evaluated how the de-regulated BRG1 expression affect the binding of activators and repressors on the NANOG promoter. We found 4 E2F binding motifs on NANOG promoter, which can be occupied by RB1 and RB2/P130. These are members of the retinoblastoma gene family. In MSC with a silenced BRG1, the relative binding of the 2 retinoblastoma proteins increased, and this was associated with the recruitment of DNMT1. This induced the methylation of CpG on the NANOG promoter. Opposingly, when a high level of BRG1 was present, the same E2F binding motifs were docking sites for BRG1, which induced chromatin compaction without CpG methylation but with increased histone deacetylation, associated with the presence of HDAC1 on E2F binding sites. Besides the sharp regulation of the NANOG expression, we evidenced, through proteomic analysis, that the de-regulation of the SWI/SNF function affected the expression of histones and other nuclear proteins involved in “nuclear architecture,” suggesting that BRG1 may act as global regulator of gene expression.</p