Oxidative Stress, Bone Marrow Failure, and Genome Instability in Hematopoietic Stem Cells

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease

Double-Strand Break Repair by Interchromosomal Recombination: An <i>In Vivo</i> Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

<div><p>Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G²). However, no <i>in vivo</i> model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-<i>Sce</i>I endonuclease, and a tetracycline-inducible I-<i>Sce</i>I endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-<i>Sce</i>I expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells <i>in vivo</i> search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation. </p> </div

DSB-induced GFP+ recombinants in hematopoietic subpopulations isolated from bone marrow of GS mice.

<p>Colonies were scored by inverted fluorescent microscopy and faint background fluorescence of negative controls was subtracted out of total repair frequency. Representative phase contrast and fluorescent microscopy images of GFP+ recombinants from bone marrow CFC assay. Granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM), Granulocyte-macrophage (CFU-GM), Granulocyte (CFU-G), Macrophage (CFU-M), Pre-B cell (Pre-B), and Burst forming unit-erythroid (BFU-E). Magnification 400X.</p

Quantitative analysis of GFP+ cells in all mice analyzed.

<p>The number of GFP+ cells in each organ of analyzed mice was determined. Establishment of gates is described in text. From FACS analysis, the average number of GFP+ recombinant cells per million cells and the standard deviation of each was calculated for seven organs and represented in bar graph form. Negative controls are shown in red bars (n=8), and +TET are shown in green bars (n=47). H=heart, P=pancreas, Li=liver, K=kidney, S=spleen, Lu=lung, T=thymus. Organs with statistically significant increased numbers of GFP+ cells groups are indicated by ** above the error bars.</p

Quantitative analysis of GFP+ cells by age.

<p>(<b>A</b>) In the young mouse cohort (age<5.5 months), negative controls are shown in red bars (n=5), and +TET are shown in grey bars (n=16). Organs with statistically significant increased numbers of GFP+ cells groups are indicated by ** above the error bars. (<b>B</b>) In the older mouse cohort (age>8 months), negative controls are shown in red bars (n=11) and +TET are shown in black bars (n=31). (<b>C</b>) Comparison of +TET young mice (grey bars) versus +TET old mice (black bars) from A and B. Organs with statistically significant different numbers of GFP+ cells by age are indicated by ** above the error bars. For all panels, H=heart, P=pancreas, Li=liver, K=kidney, S=spleen, Lu=lung, T=thymus.</p

Analysis of GFP+ MEFs post-TET.

<p>(<b>A</b>) Phase contrast and matched fluorescent microscopy images of MEFs in culture—magnification 400X. Top row-untreated MEFs. Second row-96 hrs post-electroporation with I-<i>Sce</i>I expression plasmid. Third and fourth rows-96 hrs after addition of tetracycline to the culture medium (+TET). (<b>B</b>) Representative FACS plot of MEFs with GFP positivity in log scale on the x axis plotted against number of cells on the y axis. Upper plot--untreated MEFs. Lower plot-- +TET treated MEFs. In this sample, the GFP+ population is 12.4%. (<b>C</b>) Confirmation of GFP+ cells after FACS single cell sorting for GFP+ MEFs. Cells within the GFP+ gate indicated in B lower panel were sorted and then viewed by fluorescent microscopy—magnification 400X.</p

Structure and confirmation of the 1S and 2S GFP transgenes.

<p>(<b>A</b>) For each construct schematic, the numbers of bases are indicated to show the lengths of homology between the two as well as the relative positions of the engineered I-<i>Sce</i>I restriction sites. The 3’UTR sequences of the two constructs do not share homology and are indicated as a hatched box of 1270 bp for 1S-GFP and a grey box of 535 bp for 2S-GFP; these non-homologous sequences allow for PCR amplification specific to each transgene. Nested PCR primer pairs used for verification of intact construct sequences and for analysis of GFP+ hematopoietic colonies are indicated. Primers 1F-4R followed by 2F-3R amplify sequence flanking the I-<i>Sce</i>I site in 1S-GFP. Primers 1F-7R followed by 5F-6R amplify sequence flanking the I-<i>Sce</i>I site in 2S-GFP. (<b>B</b>) Southern blotting to estimate copy number utilized a GFP ORF DNA fragment of 3.1 kb and diluted to pg amounts that approximated 0, 0.2, 1.0, and 5.0 copies per genome spiked into 10µg non-transgenic mouse DNA. Genomic DNA from single transgenic mice (either 1S-GFP or 2S-GFP) was digested with restriction endonucleases within the GFP promoter and ORF of both transgenes to yield a 3.1 kb fragment. Band intensities are consistent with 4-5 copies of 1S-GFP and 2-4 copies of 2S-GFP, and were confirmed with Q-PCR data on the same samples (data not shown). (<b>C</b>) PCR reactions flanking each DSB site in the two GFP constructs confirm intact I-SceI recognition sites. Nested PCR as described in Materials amplified each transgene shown in the left side lane of each image. Digestion with I-<i>Sce</i>I endonuclease produced the expected sizes indicated in the middle lane of each image. Right side Marker lane PhiX.</p

Structure and confirmation of the tetracycline inducible I-<i>Sce</i>I transgene.

<p>(<b>A</b>) For details of the bicistronic I-<i>Sce</i>I transgene construct refer to [65]. (<b>B</b>) MEFs derived from GS mice were cultured in media supplemented with TET at 2μg/mL for 48 hours. Total protein extracts were harvested and analyzed by Western blotting. By 48 hours post-TET, detectable quantities of I-<i>Sce</i>I endonuclease can be observed. As a negative controls, total protein extracts were harvested from cultured E14 ES cells or uncultured MEFs from GS mice. Loading control: Western blotting for β-actin.</p