Low Dose-Radiation of Bone Marrow Stromal Cells Supports Pre-Leukemic Cell Survival Via Adhesion and Inflammatory Signaling Stimulation

Abstract 4610

Low-dose irradiation (LDI) exposure is a form of environmental carcinogen, which is of significant interest after the nuclear accident in Japan. In this study, we investigated the LDI-induced molecular alterations of bone marrow (BM) –derived stromal cells (MSCs), and the biological response of pre-leukemic cells neighbor to LDI exposed MSCs. As the model of pre-leukemic cells, we utilized the Epstein- Barr virus (EBV) infected and immortalized B lymphocyte cell line (EBV-B).

In MSCs, exposed to 100 mGy irradiation (4MVX rayfrom a LINAC) caused cell growth inhibition (75.8±2.4 % of control, p<0.001, MTT assay) with moderate apoptosis induction (SubG1 %; control 2.3±0.4, radiation 7.3±2.3, p=0.03, PI cell cycle analysis) at 24 hrs. Screening up to 28,869 genes by cDNA microarray (Human Gene 1.0 ST Array, Affymetrix) revealed 48 up-regulated and 45 down-regulated genes (>1.3 fold) in irradiated MSCs (at 24 hrs) compared to non-irradiated MSCs. The functional network analysis of cDNA microarray data by MetaCore (GeneGo) showed an enrichment of the adhesion/ECM remodeling pathways. TaqMan RT-PCR confirmed significant up-regulation of ECM scaffold Sulfatase1 mRNA (2.0±0.1 fold, p=0.004). Proteomic analysis utilized isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems), identified 32 up-regulated and 1 down-regulated proteins (p<0.05) among 1,536 detected proteins comparing irradiated MSCs (at 24 hrs) with non-irradiated MSCs. KEGGontology analysis (Kyoto University) found the activation of focal adhesion and apoptosis pathways in irradiated MSCs; among 32 up-regulated proteins, six are involoved in adhesion/cytoskeleton regulation (Prelamin-A; p<0.001, Catenin beta-1; p=0.03, Keratin type II; p=0.02, Collagen alpha1; p=0.02, alpha actinin1 and 4; p=0.01, p=0.02) and three are involved in apoptosis/senescence (Stress-70 protein l; p=0.02, Endoplasmin; p=0.008, Prohibitin; p=0.04). These changes were accompanied with increased secretion of inflammation and coagulation marker protein plasminogen activator inhibitor 1 (PAI-1) from irradiated MSCs (control 10.1±0.03 ng/mL, radiation >15.0 ng/mL, at 72hrs). We then investigated the effect of LDI exposed MSCs on EBV-B cell proliferation. Under low-serum growth condition (1% FBS), co-culture with pre-irradiated (100 mGy) MSC layers supported EBV-B cell proliferation more prominently than non-irradiated MSCs (118.3±4.0 % viable cell number, p<0.001, 72hrs). Gene expression profiles by cDNA microarray and sequenced MetaCore ontology revealed 53 up-regulated and 39 down-regulated genes showing an enrichment of the coagulation and cell adhesion pathways in EBV-B cells co-cultured with pre-irradiation of MSCs compared to the ones co-cultured with non-irradiated MSCs. Up-regulation of anti-apoptotic BCL2 mRNA (0.5±0.03 fold, p=0.02) and inflammatory IL8 mRNA (2.0±0.03 fold, p<0.001) was further detected by TaqMan RT-PCR. The purity of EBV-B cells separated from MSCs was confirmed by lack of CD90 mRNA expression by PCR. These data suggest that LDI exposure to MSCs leads to latent adhesion and inflammatory signaling in MSCs, which potentially activates pro-survival pathways of co-cultured pre-leukemic EBV-B cells. In summary, we present the biosignature of the response of BM-derived stromal cells to LDI utilizing gene array and quantitative proteomics analysis that may have important implications for risk assessment in environmental medicine.