ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells

Abstract

Background: Unrepaired DNA double-stranded breaks (DSBs) cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ) pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID) and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms.

Results: Here we investigate roles for the NHEJ factor ARTEMIS in multipotent mesenchymal stem/progenitor cells (MSCs), as putative sarcomagenic cells of origin. We demonstrate a key role for ARTEMIS in sarcoma suppression in a sensitized mouse tumor model. In this context, we found that ARTEMIS deficiency led to chromosomal damage but, paradoxically, enhanced resistance and proliferative potential in primary MSCs subjected to various stresses. Gene expression analysis revealed abnormally regulated stress response, cell proliferation, and signal transduction pathways in ARTEMIS-defective MSCs. Finally, we identified candidate regulatory genes that may, in part, mediate a stress-resistant, hyperproliferative phenotype in preneoplastic ARTEMIS-deficient MSCs.

Conclusions: Our discoveries suggest that Art prevents genome damage and restrains proliferation in MSCs exposed to various stress stimuli. We propose that deficiency leads to a preneoplastic state in primary MSCs and is associated with aberrant proliferative control and cellular stress resistance. Thus, our data reveal surprising new roles for ARTEMIS and the NHEJ pathway in normal MSC function and fitness relevant to tumor suppression in mesenchymal tissues.

Figure 1

Art deficiency accelerates mesenchymal tumor development. (a) Tumor-free survival analyses of Art^Δ/Δ Trp53^Δ/+ mice (red circles) versus Trp53^Δ/+ mice (blue, diamond). Plotted is the surviving fraction of those mice that developed tumors as a function of time (weeks). Significance was determined by t-testing. (b) Hematoxylin and eosin (H&E) staining of an anaplastic sarcoma (left) and a chondrosarcoma (center) found in Art^Δ/Δ/Trp53^Δ/+ mice, shown at ×10 and ×40 magnification. Normal bone marrow from an Art mouse (right) is shown for comparison. Boxed areas in ×10 magnification demarcate regions shown in ×40 magnification. Lines represent scale bars (200 μm in ×10 magnification; 50 μm in ×40 magnification). (c) Spectral karyotype (SKY) analysis of Art^Δ/Δ Trp53^+/Δ osteosarcoma. Shown are the 4',6'-diamidino-2-phenylindole (DAPI) stained metaphase (inverted image, top left) with superimposed chromosome contours (blue), spectral image of SKY painted metaphase spread (top, middle), and computer classified image (top, right), as well as the karyotype table showing approximate hyperdiploidy (bottom).

Figure 2

Art prevents chromosome instability in MSCs. (a) SKY analysis of wild type (WT) (top) or Art^Δ/Δ (bottom) mesenchymal stem cells (MSCs). An example of a chromatid break, typifying damage in Art^Δ/Δ cells, is indicated by an arrow and shown magnified. (b) Summary of spontaneous chromosomal abnormalities in WT versus Art^Δ/Δ. (c) Distribution of chromosome number in WT versus Art^Δ/Δ MSC karyotypes. Shown is the percentage of metaphase spreads from each karyotype harboring the indicated number of chromosomes.

Figure 3

Art-deficient MSCs differentiate normally. (a) Art^Δ/Δ and WT MSCs were grown in adipogenic culture medium for 7 days, fixed and stained with the fluorescent lipid-binding dye LipidTOX (Invitrogen). Shown are bright field, fluorescent and merged images for each. (b) Art^Δ/Δ and WT MSCs were grown in osteogenic culture medium for 14 days, fixed and stained with Alizarin red to detect mineralization indicative of osteocytic development. (c) Art^Δ/Δ and WT MSCs were grown in adipogenic or unsupplemented culture medium for 14 days, fixed and stained with LipidTOX and DAPI counterstain. The fraction of LipidTOX-positive cells was determined for each sample and culture condition at days 0, 7 and 14. Error bars indicate standard error. (d-f) Mitotic indices for undifferentiated, adipogenic or osteogenic cultures of Art^Δ/Δ and WT MSCs were determined by immunostaining for M-phase marker phosphorylated histone H3 (phospho-H3). (d) Representative fluorescence micrographs of phospho-H3-positive cells (green), DAPI DNA counterstain (blue), and merged are shown for Art^Δ/Δ and WT MSCs. Scale bars, 10 μm. Fractions of positive phospho-H3 staining were determined at days 0, 3, 7 and 14 of (e) undifferentiated control, (f) adipogenic or (g) osteogenic culture conditions. Error bars indicate standard error.

Figure 4

Art is dispensable for MSC resistance to ionizing irradiation. (a) Three independent biological replicates of WT or Art^Δ/Δ MSCs or control fibroblasts were plated at 5 × 10⁴ cells per dish each and cultured for 10 days. Viable cell counts, measured by exclusion of the vital dye trypan blue, were determined every 2 days. Error bars indicate standard error. (b) Clonogenicity assay for sensitivity to ionizing irradiation. A total of 1 × 10⁵ WT or Art^Δ/Δ MEFs or 5 × 10⁵ WT or Art^Δ/Δ MSCs were irradiated at the indicated doses, plated to 100-mm dishes and cultured until colonies were visibly evident for the unirradiated WT controls of each cell type. Cells on all plates were then fixed and stained with crystal violet histological stain. (c) Quantification of clonogenicity (from (b)) following IR. Colonies were counted and normalized to the unirradiated control for MEFs or MSCs, respectively. Error bars indicate standard error. (d) WT and Art^Δ/Δ MSCs or control fibroblasts were exposed to ionizing radiation (IR) at indicated doses, plated at equivalent densities in triplicate and harvested for analysis after 7 days. Viable cell counts were determined for single-cell suspensions by trypan blue exclusion. Relative resistance to IR is expressed as the viable cell count at each dose normalized to the unirradiated control for each sample. Error bars denote standard error.

Figure 5

Art modulates cell cycle response following IR. (a) Representative micrographs of WT and Art^Δ/Δ MSC metaphase spreads. (b) The mitotic indices of WT (filled bars) and Art^Δ/Δ (open bars) MSCs following exposure to IR at indicated doses were determined by quantification of metaphase cells. Mitotic index is expressed as the percentage of mitotic figures per total nuclei. Significance was determined by t-testing (**P < 0.01; ***P < 0.005). (c-g) The mitotic indices of WT and Art^Δ/Δ MSCs or control fibroblast 6, 12 and 24 hours following 1 Gy ionizing irradiation were determined by immunostaining for the mitotic marker pH3. (c) Shown are representative merged micrographs of pH3+ (green) and DAPI DNA counterstained (blue) WT or Art^Δ/Δ MEFs and MSCs at each time point after IR. Scale bars, 10 μm. The fraction of phospho-H3-positive cells for WT (filled bar) and Art^Δ/Δ (open bar) MSCs (d) or MEFs (f) were determined for each time point after IR. Data from (d and f) were also normalized to the 0 Gy controls. Normalized data are shown for MSC (e) and MEF (g). Error bars indicate standard error.

Figure 6

Art-deficient MSCs are resistant to culture stress by serum withdrawal. (a) Light micrographs of Art^Δ/Δ or WT MSCs exposed to serum-free (serum starvation) versus normal (10%) serum (control) culture conditions. Cell cultures were photographed after either 2 or 6 days of serum withdrawal. (b) Art^Δ/Δ and WT MSCs after culture in normal conditions for 6 days. (c) Viability of WT (filled bars) versus Art^Δ/Δ (open bars) MSCs after culture in normal (control) or serum withdrawal (serum starved) conditions for 7 days. Viable cell counts were determined as the number of trypan blue-excluding cells normalized to the normal serum control. (d) Fold reduction in survival of WT (filled bars) or Art^Δ/Δ (open bars) following 7 days of serum withdrawal. Significance in all assays was determined by t-testing (***P < 0.005).

Figure 7

Art^Δ/Δ MSCs show an attenuated transcriptional response to serum withdrawal. (a) Schematic showing experimental design for comparative gene expression analysis. RNA was isolated from duplicate cultures representing either WT (blue) or Art^Δ/Δ (red) MSCs cultured in either normal (solid line with filled nuclei) or serum starvation (dashed line with open nuclei) media. All samples were analyzed by hybridization to Affymetrix GeneChip Mouse 430 2.0 microarrays. Relative fold change (RFC) in transcription levels was determined for serum-starved cells versus corresponding controls in WT and in Art^Δ/Δ samples. Using a threshold of threefold or greater RFC, WT and Art^Δ/Δ data were comparatively analyzed and results were categorized as unique to WT cells (blue), unique to Art^Δ/Δ cells (red) or common to both (overlap). In total, 157 genes were identified with a threefold or greater RFC in WT or Art^Δ/Δ or both. Number of genes identified in each category is indicated on the Venn diagram. (b) RFC data for each of the 157 genes in (a). Shown are RFC data for each gene in WT cells (blue bars) and in Art^Δ/Δ cells (red bars). Negative RFC values indicate lower expression in serum-starved cells relative to control; positive RFC values indicate elevated expression in serum-starved cells relative to controls. (c) RFC for genes common to both WT and Art^Δ/Δ cells from (a). Plotted are RFC for WT (blue) and Art^Δ/Δ (red) cells as in (b).

Figure 8

Deregulation of stress-response, proliferation and differentiation pathways in serum-starved Art^Δ/Δ MSCs. (a) Difference in RFC (ΔRFC) between WT and Art^Δ/Δ cells (defined as [WT RFC] - [Art^Δ/Δ RFC]) was determined for the 157 genes identified in Figure 7a. Plotted are data for all genes showing ΔRFC = 2 or greater. Positive ΔRFC values indicate a higher RFC in WT than in Art^Δ/Δ samples; conversely, negative ΔRFC values denote lower RFC in WT than in Art^Δ/Δ samples. Individual gene names are indicated. (b) RFC for genes in (a) are indicated, with WT (dark fill) and Art^Δ/Δ (light fill) data overlaid. Genes with gene ontology (GO) annotations in BMP/WNT signaling, or in other growth factor signaling, are indicated by red/green (WT/Art^Δ/Δ) or blue/orange (WT/Art^Δ/Δ) shading, respectively. (c-e) RFC data for genes with GO annotations for stress response (c), cell proliferation (d), or cell differentiation (e) are shown. RFC for each gene in WT (gray bars) and Art^Δ/Δ (open bars) samples are overlaid. Individual gene names are indicated. Genes with in BMP, WNT or growth factor signaling pathways are highlighted in red.