Integrative Genomic and Proteomic Analysis Of Low-Dose ionizing Irradiation Effects On Bone Marrow Stromal Microenvironment and On Survival Of Pre-Leukemic Cells
Authors: Tabe Yoko , Jin Linhua , Hatanaka Yasuhito , Matsushita Hiromichi , Kazuno Saiko , Fujimura Tsutomu , Ueno Takashi , Sasai Keisuke , Iwabuchi Kazuhisa , Andreeff Michael , Konopleva Marina , Miida Takashi 
Published: October 2019

Abstract
Ionizing radiation is known to induce remodeling of stromal microenvironment and enhance cancer progression. In this study, we investigated the molecular alterations of low-dose ionizing radiation (LDIR) induced non-targeted/bystander responses which affect a complex interplay of stromal cells and pre-leukemic cells in the bone marrow (BM) microenvironment.
As a model of BM stromal cells and pre-leukemic cells, we utilized primary BM-derived stromal cells (MSCs) and the Epstein- Barr virus (EBV) infected and immortalized pre-leukemic B-lymphocyte cell line (EBV-B). LDIR (100 mGy, 4MV X ray from a LINAC) caused cell growth inhibition and moderate apoptosis induction in MSCs (viable cells % of control; 75.8 ± 2.4, specific SubG1 % 7.1 ± 0.8 at 24 h) but not in EBV-B cells. We further observed persistent upregulation of p21 mRNA (p< 0.001, RQ-PCR) after acute low-dose irradiation in MSCs but not in EBV-B cells, suggesting radiation induced senescence-like changes in MSCs. In EBV-B cells co-cultured with MSCs, low-dose irradiation induced moderate cell growth inhibition (viable cells % of control; 81.3 ± 6.5) without significant apoptosis induction.
To gain insights into the molecular changes induced by LDIR in both, MSC and EBV-B cells we utilized genomic and proteomic analyses. To exclude possible contamination of MSCs, we confirmed negative expression of CD90 mRNA in the tested EBV-B samples. We first screened up to 28,869 genes by cDNA microarray (Affymetrix) and performed functional network analysis by MetaCore (GeneGo). LDIR induced upregulation of 48 genes and the downregulation of 45 (i.e., > 1.3-fold regulation) with prominent stimulation of cell adhesion pathways in MSCs. Of note, 31 of 48 up-regulated genes were small nucleolar RNAs. In EBV-B cells, LDIR upregulated 69 genes/downregulated 130 genes with significant stimulation of the TGFbeta dependent induction of epithelial-mesenchymal transition (EMT) pathways. In EBV-B cells co-cultured with MSCs, LDIR induced immune response signaling along with integrin-mediated cell-matrix adhesion pathway with 42 genes upregulation / 34 genes downregulation. Up-regulation of inflammatory IL8 mRNA (2.0±0.03 fold, p<0.001) by LDIR were further detected in EBV-B cells co-cultured with MSCs. Since ionizing radiation is known to change levels of specific microRNA depending on cell type, we investigated the changes of four microRNA, let-7a, miR-16, miR-19b and miR-21 by RT-PCR. After acute LDIR, all of the tested miRNAs were upegulated in EBV-B cells cultured alone, but downregulated in EBV-B cells co-cultured with MSCs. These changes were accompanied by the coordinate modulation of their common target GPAM mRNA, a key enzyme of phospholipids, and ribozyme CPEB3 mRNA. Indeed, proteomic analysis by isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems) detected the upregulation of Protein kinase C delta (p = 0.04) which is strictly dependent on the presence of phospholipids. iTRAQ detected more than 1500 proteins in MSCs and EBV-B cells. In MSCs LDIR resulted in 32 upregulated proteins and 1 downregulated protein (p < 0.05) with the activation of focal adhesion (6 proteins, p < 0.001) and apoptosis/senescence pathways (three proteins, p = 0.02), concordant with cDNA microarray data. In EBV-B cells, LDIR induced upregulation of 47 proteins including 19 ribosome related proteins (40%) and downregulation of 19 proteins including 6 apoptosis/senescence related proteins, suggesting an activation of molecular repair mechanisms from LDIR-triggeredstress. In contrast, under MSC co-culture conditions LDIR downregulated 25 of ribosomal proteins (74% of the 34 downregulated proteins) in EBV-B cells, indicating suppression of the ribosomal biogenesis and translational activity, which might cause the observed cell quiescence with cell cycle arrest of EBV-B cells mediated by the low-dose irradiated neighboring MSCs.
In summary, we have demonstrated the LDIR effects on BM-derived stromal cells and pre-leukemic cells under MSC co-culture conditions mimicking the BM microenvironment. We conclude that LDIR may support pre-malignant cells survival via direct activation of TGFbeta dependent EMT pathways, which contribute to antiapoptosis, and via interactions with irradiated neighboring BM stromal cells which promote quiescence and survival of pre-leukemic cells.

Disclosures:
No relevant conflicts of interest to declare.