Chk1 haploinsufficiency results in anemia and defective erythropoiesis

Abstract

Background: Erythropoiesis is a highly regulated and well-characterized developmental process responsible for providing the oxygen transport system of the body. However, few of the mechanisms involved in this process have been elucidated. Checkpoint Kinase 1 (Chk1) is best known for its role in the cell cycle and DNA damage pathways, and it has been shown to play a part in several pathways which when disrupted can lead to anemia.

Methodology/principal findings: Here, we show that haploinsufficiency of Chk1 results in 30% of mice developing anemia within the first year of life. The anemic Chk1+/- mice exhibit distorted spleen and bone marrow architecture, and abnormal erythroid progenitors. Furthermore, Chk1+/- erythroid progenitors exhibit an increase in spontaneous DNA damage foci and improper contractile actin ring formation resulting in aberrant enucleation during erythropoiesis. A decrease in Chk1 RNA has also been observed in patients with refractory anemia with excess blasts, further supporting a role for Chk1 in clinical anemia.

Conclusions/significance: Clinical trials of Chk1 inhibitors are currently underway to treat cancer, and thus it will be important to track the effects of these drugs on red blood cell development over an extended period. Our results support a role for Chk1 in maintaining the balance between erythroid progenitors and enucleated erythroid cells during differentiation. We show disruptions in Chk1 levels can lead to anemia.

Figure 1. Chk1 is expressed in erythroid progenitor cells.

(A) Expression profile of Chk1 in the hematopoietic cell types from a previous study . Erythrocytes, granulocytes, and LT-HSCs were isolated from WBM. Erythroid Progenitors were Ter-119⁺, CD3⁻,CD4⁻,CD8⁻,Mac-1⁻,Gr-1⁻, and B220. LT-HSCs were side-population (SP)⁺, Sca-1⁺, c-Kit⁺, and Lin⁻ (Mac-1, Gr-1, Ter119, B220, CD4, CD8) (B) Gross morphology of spleens from a WT and Chk1 mouse are drastically different. (C) Spleen weights of WT and anemic Chk1+/−. (D.) 100,000 WBM cells were plated in Methocult specific for erythroid colony formation. BFU-E and CFU-E were counted 6 days after plating and a lack of BFU-E formation was observed from the Chk1+/− cells.

Figure 2. Chk1+/− mice show a breakdown of hematopoietic tissue structure with age.

(A) H&E stained WT and Chk1+/− spleen sections at 5× and 40× magnification and images scaled at 50 µm. Disruption of spleen architecture is plainly visible. Also shown are sections of spleen from a 52-week-old WT and anemic Chk1+/− mouse stained with Ter-119 (HPR, brown, 5×) or F4/80 (HPR, brown, 40×) and counterstained with hematoxylin. Chk1+/− mice show a breakdown in spleen architecture and a massive increase in histiocytes. (B) WT and Chk1+/− spleens stained with F4/80 under 60× magnification and images scaled at 20 µm. (C) H&E sections (20×) of sternum from a WT and Chk1+/− mouse showing a loss of bone marrow architecture with a colossal histiocyte invasion. F4/80 (HPR, brown, 40×) stained sternum sections shows an overwhelming increase in histiocytes compared to WT.

Figure 3. A significant number of Chk1+/− mice become anemic with age.

(A) Kaplan-Meier plot showing the incidence of anemia in the Chk1+/− mice compared to WT mice. A log-rank (Mantel-Cox) test was used to measure significance (p<0.05). Complete blood counts were done on 52-week-old WT and anemic Chk1+/− mice and significance was established by a student's t-test. (B) White blood counts (WBC), (C) platelet counts, and (D) red blood cell parameters (red blood cell counts, (RBC), hemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are shown. A student's t-test was used to determine significance; an asterisk indicates a p<0.05.

Figure 4. Anemic Chk1+/− mice exhibit severe defects in several stages of erythropoiesis.

(A) Representative flow cytometry plots showing CD71 and Ter-119 staining used to separate the stages of erythropoiesis for WT and Chk1+/− mice. (B) The MEP and stages I–IV of erythropoiesis were analyzed via flow cytometry from the whole bone marrow of anemic Chk1+/− mice and 52-week-old WT mice. A student's t-test was used to determine significance; an asterisk indicates a p<0.05.

Figure 5. Chk1+/− erythroid progenitors show a marked increase in spontaneous DNA damage foci.

Progenitors from stages I–IV were sorted from WT and Chk1+/− mice and immunostained with DNA damage markers 53BP1(green), pH2AX(red) and DAPI(blue). The Chk1+/− erythroid progenitors from stages I–IV showed dramatic increase and co-localization of 53BP1 and pH2AX at the site of spontaneous DNA damage and double strand break in the nucleus, resulting in miscoordination of enucleation during erythropoiesis compared to WT counterparts at 60× magnification and images scaled at 20 µm.

Figure 6. Chk1+/− mice show a disruption in enucleation of erythrocytes in the bone marrow.

(A) Representative flow cytometry plot of enucleation assay used. Syto 16 is cell permeable, whereas Sytox Blue is cell impermeable, thus the two dyes in tandem allow the separation of enucleated and nucleated erythrocytes . Low FSC Ter-119+ cells or high FSC Ter-119+ were examined for the percentage of nucleated or enucleated cells. Low FSC (small) cells are the more mature erythrocytes, while high FSC (large) cells are the more immature erythrocytes. (B) The P1 gated cells, which should almost entirely consist of RBCs, have fewer properly enucleated erythrocytes with an increase in cells having their nuclei in Chk1+/− whole bone marrow. The P2 gated cells, the majority of which should be immature erythrocytes with their nuclei still intact show a decrease in nucleated cells with a concurrent increase in enucleated cells in the Chk1+/− whole bone marrow. (C) Progenitors from stages I–IV were sorted from WT and Chk1+/− mice and immunostained with Alexa-flour 594 Phalloidin(red) and DAPI(blue). The WT erythroid progenitors from stages I–IV show dynamics of actin filaments during CAR in erythropoiesis. WT erythroid progenitors in Stages III and IV display formation of CAR (yellow arrows). Aberrant CAR formation (yellow arrows) is observed in stages III and IV Chk1+/− erythroid progenitors at 60× magnification and images scaled at 20 µm.

Figure 7. Chk1 expression in human erythroid progenitors and anemia patients.

(A) Taqman real-time PCR was used to examine the expression levels of Chk1 in human erythroid progenitors and 18s was used as an internal control (n = 3). (B) A decrease in Chk1 levels (one probe) was observed in patients (individual patients are represented as dots and the graph) with refractory anemia with blasts when compared to healthy individuals (Z-test, p<0.05; GEO DataSets ID = GDS2118; Affy probe ID = 238075_at) . GEO DataSets were searched for the key words anemia and human via the NCBI website (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gds). The results were further filtered to select for studies using hematopoietic progenitors and examining types of anemia. Studies utilizing samples from transplant patients, cancer patients, or malaria patients were discarded. Only one study met these criteria.