Deletion of PTPN1 Promotes the Development of Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 795-795
Author(s):  
Fatoumata Jobe ◽  
Bhumika Patel ◽  
Hideki Makishima ◽  
Bartlomiej P Przychodzen ◽  
Robert E Hutchison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Tyrosine Phosphatase ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Obesity And Diabetes

Abstract Deletion of chromosome 20q [del(20q)] is a common chromosomal abnormality associated with myeloid neoplasms including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN overlap disorders and acute myeloid leukemia (AML). The del(20q) lesion is often associated with myeloproliferative features; it is present in patients with myelofibrosis (MF) at a high frequency (24%) and thus considered to be one of the most frequent cytogenetic abnormalities in MF (Wassie et al., Br J Haematol. 2015). The del(20q) lesion can also coexist with JAK2V617F mutation in MPN/MF. However, the target tumor-suppressor gene(s) within chromosome 20q involved in the pathogenesis of MF remains unknown. The PTPN1 locus maps to human chromosome 20q13.1-q13.2. PTPN1 (also known as PTP1B) is a ubiquitously expressed non-receptor tyrosine phosphatase that has been linked to metabolism and cancer. Mice deficient in Ptpn1 exhibit resistance to diet-induced obesity and diabetes. Both oncogenic and tumor suppressor functions for PTPN1 have been suggested. PTPN1 can negatively regulate the JAK/STAT signaling, which is frequently found activated in MPN. Here, we report the identification and functional consequences of PTPN1 deletion in the pathogenesis of MF. Deletion of PTPN1 was identified in 14% cases of MF. Conditional deletion of Ptpn1 in the mouse hematopoietic compartment resulted in significant increases in white blood cell and neutrophil counts in the peripheral blood and enlargement of spleen size. Flow cytometric analyses showed significant expansion of myeloid (Gr-1+/Mac-1+) precursors in the bone marrow (BM) and spleens of Ptpn1-deleted mice compared with control animals. Megakaryocytic (CD41+/CD61+) precursors were also significantly increased in the spleens of Ptpn1-deleted mice. Flow cytometric analyses also revealed significant increases in absolute numbers of LSK cells (Lin-Sca1+c-kit+) and its subsets including long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multi-potent progenitors (MPP) in the spleens of Ptpn1-deleted mice. Hematopoietic progenitor colony assays showed significant increases in myeloid (CFU-GM) and megakaryocytic (CFU-Mk) colonies in the BM of Ptpn1-deleted mice compared with control mice BM. Histopathologic analysis demonstrated fibrosis (grade 2) in the BM and spleens of Ptpn1-deleted mice at 52 weeks after induction, whereas control animals did not exhibit fibrosis at that age. Together, these results suggest that deletion of Ptpn1 induces an MPN-like phenotype, which progresses to MF over time. Moreover, transplantation of Ptpn1-deficient BM into lethally irradiated wild-type animals resulted in fibrosis at 18 weeks after transplantation, demonstrating that the effect of Ptpn1 loss in the development of myelofibrosis is cell-intrinsic. Competitive repopulation assays using BM from control or Ptpn1-deficient (CD45.2+) mice with wild type congenic (CD45.1+) mice showed that deletion of Ptpn1 enhances the repopulation capacity of hematopoietic stem cells. Biochemical analyses revealed that depletion of Ptpn1 enhanced JAK2/STAT5, AKT and ERK signaling in the BM of Ptpn1-deleted mice. Furthermore, we observed that deletion of Ptpn1 in Jak2V617F knock-in mice accelerates the development of myelofibrosis. In conclusion, our results establish a tumor-suppressor function for PTPN1 in MF. Disclosures No relevant conflicts of interest to declare.

scholarly journals Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1174-1174
Author(s):  
Darlene Monlish ◽  
Angela Herman ◽  
Molly Romine ◽  
Sima Bhatt ◽  
Laura G. Schuettpelz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Immune Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Tlr2 Agonist ◽  
Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM3CSK4, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM3CSK4 led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2-/- + Tlr2+/+ bone marrow transplanted into Tlr2+/+ or Tlr2-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM3CSK4, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM3CSK4-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

Angiopoietin-Like 3 Affects Homing Ability of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2632-2632
Author(s):  
Masato Umikawa ◽  
Junke Zheng ◽  
HoangDinh Huynh ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Physiological Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Intrinsic Effect

Abstract Abstract 2632 Angiopoietin-like proteins (Angptls) are a seven-member family of secreted glycoproteins that share sequence homology with angiopoietins. It is known that several members of the Angptl family including Angptl3 support ex vivo expansion of hematopoietic stem cells (HSCs). However, the physiological role of Angptls in the hematopoietic system is not well known. Here we show that Angptl3 is expressed by both bone marrow stromal cells and HSCs. To study the intrinsic effect of Angptl3 in mouse HSCs, we isolated the same number of HSCs from wild-type and Angptl3-null mice and performed reconstitution analysis. Adult bone marrow Angptl3-null HSCs showed decreased repopulation compared to wild-type HSCs, suggesting that Angptl3 has cell-autonomous effect on HSC activity. By contrast, HSCs isolated from liver of the null mice had enhanced HSC repopulation activity than their wild-type counterparts. To study whether this effect is caused by difference in homing, we injected CFSE labeled wild-type HSCs and Angptl3 null HSCs into lethally irradiated mice, and checked the homing to bone marrow, spleen, and liver. While homing of these two types of cells to bone marrow or spleen was not significantly different, Angptl3 null HSCs homed better to the liver than the wild-type HSCs. Our result suggests that Angptl3 is important for the retention of HSCs in the bone marrow, and the absence of Angptl3 leads HSCs to move to extramedullary organs such as liver. Disclosures: No relevant conflicts of interest to declare.

Deletion of Bak and Bax in Tie2+ BM Hematopoietic Stem Cells Induces a B Cell Lymphoproliferative Disorder.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1247-1247
Author(s):  
Phuong L. Doan ◽  
J. Lauren Russell ◽  
Sarah K. Meadows ◽  
Heather A. Himburg ◽  
Pamela Daher ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Lymphoproliferative Disorder ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Targeted Deletion

Abstract Abstract 1247 Poster Board I-269 Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal, mature B-cell lymphocytosis, lymphadenopathy and splenomegaly. The pathogenesis of CLL is thought to be related to abnormal programmed cell death mechanisms but the precise contribution of several pro-apoptotic genes remains unknown. Elucidation of the pathogenesis of CLL is also limited by the lack of animal models which reproduce the CLL phenotype. We present a transgenic mouse model in which conditional, tissue-specific deletion of Bax is achieved in Tie2+ hematopoietic stem cells (HSCs) via Cre-LoxP recombination. In this model, Tie2Cre;Bak-/-;BaxFl/- mice carried constitutive deletion of Bak and targeted deletion of Bax in Tie2+ BM HSCs. Surprisingly, the combined deletion of Bak and Bax in Tie2+ cells caused a pronounced leukocytosis with total WBCs>100,000 in 8-week old mice. In contrast, Tie2Cre;Bak-/-;BaxFl/+ mice, which retained one Bax allele, had normal peripheral blood WBCs comparable to wild type C57Bl6 mice (range 4-10,000;p<0.01 and p<0.01). Tie2Cre;Bak-/-;BaxFl/- mice had massive splenomegaly with a 6-fold greater spleen weight compared to Tie2Cre;Bak-/-; BaxFl/+ controls. Review of PB smears demonstrated a preponderance of “smudge” cells (70% of total cells per HPF) in the Tie2Cre;Bak-/-;BaxFl/- mice, whereas smudge cells were not observed in Tie2Cre;Bak-/-;BaxFl/+ mice. Flow cytometric analysis of PB cell differential revealed a significant increase in B cells (B220+, p=0.0001), a decrease in T cells (Thy 1.2+, p=0.0002), decreased myeloid cells (Mac1/Gr1+ cells, p=0.02), and decreased erythroid cells (Ter119+, p=0.01) compared to Tie2Cre;Bak-/-;BaxFl/- mice. The B cell-to-T cell ratio was also aberrantly increased (p=0.0007) suggesting a predominant B-cell lymphocytosis. Interestingly, while Tie2Cre;Bak-/-;BaxFl/- mice demonstrated no increase in committed BM colony forming cells (CFCs) or colony forming unit-spleen day 12 (CFU-S12), these mice contained significantly increased numbers of BM long term culture-initiating cells (LTC-ICs, p=0.04), suggesting that the combined deletion of Bak and Bax in Tie2+ cells yielded an expansion of primitive BM HSCs. Lastly, in order to confirm whether the development of the lymphoproliferative disorder was autonomous to deletion of Bak and Bax in Tie2+ HSCs, we transplanted 4 × 106 BM cells from Tie2Cre;Bak-/-;BaxFl/- mice into lethally irradiated (950 cGy) wild type B6.SJL mice. At 12 weeks post-transplant, the recipient mice were chimeric with Bak and Bax deletion in BM hematopoietic cells and Bak and Bax retained in the BM microenvironment. Remarkably, the chimeric recipient mice demonstrated a significant reduction in PB WBCs (range 23-60,000, p=0.001) and a 2.4-fold decrease in spleen size compared to Tie2Cre;Bak-/-;BaxFl/- mice (p=0.02), suggesting that the development of the lymphoproliferative disorder was modulated by Tie2+ BM endothelial cells. Taken together, these data demonstrate that the targeted deletion of both Bak and Bax, but not Bak deletion alone, in Tie2+ HSCs produces a profound lymphoproliferative disorder in mice. While it has been previously shown that constitutive deletion of Bak and Bax caused a lymphoproliferative disorder in mice (Lindsten T et al. Mol Cell 2000;6:1389), the data presented here suggest that deletion of Bak and Bax in BM HSCs may be sufficient to induce a lymphoproliferative disorder and this phenotype may be significantly modulated by the BM microenvironment. We are currently testing whether mice bearing deletion of Bak and Bax in Tie2+ cells have a monoclonal B cell leukemia and are utilizing Cre-LoxP recombination methods to determine at which point in HSC differentiation that the Bak and Bax deletions are required in order for the lymphoproliferative disorder to occur. Given the profound phenotype of the Tie2Cre;Bak-/-;BaxFl/- mice, this may be a useful animal model for the study of the pathogenesis of lymphoproliferative disorders. Disclosures No relevant conflicts of interest to declare.

Tumor Suppressor Role of HIF-1alpha in Leukemia-Initiating Cells in Flt3-ITD-Induced Myeloproliferative Neoplasm

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3578-3578
Author(s):  
Talia Velasco ◽  
Jörg Cammenga
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Molecular Response ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hypoxia-induced signalling is a major regulator in normal and malignant hematopoiesis. The transcription factor HIF-1alpha plays a crucial role in the quiescence and self-renewal of hematopoietic stem cells as well as leukemia-initiating cells (LICs) of acute myeloid leukemia and chronic myeloid leukemia. Better understanding of the requirement of the molecular response to hypoxia in LICs could lead to new therapies targeting this pathway. We have therefore investigated the effect of HIF-1alpha loss on the phenotype and biology of FLT3-ITD induced myeloproliferative neoplasm (MPN). Using a combined transgenic mouse model (Mx1-Cre; Hif-1alphafl/fl; Flt3ITD/+) we showed that deletion of HIF-1alpha leads to a more severe MPN phenotype reflected by higher numbers of white blood cells and myeloid cells in peripheral blood, as well as a more severe splenomegaly. Loss of long-term hematopoietic stem cells (LT-HSCs: Lin- Sca1+ cKit+ CD48- CD150+) and increased number of lineage-restricted progenitors (Lin- Sca1+ cKit+ CD48+ CD150-) were the most pronounced effects on a cellular level upon the loss of HIF-1alpha. The proliferative effect of the HIF-1alpha loss was cell intrinsic and not at the expense of the ability of the LICs to self-renew because the disease was transplantable into secondary recipients recapitulating the same phenotype. Mice transplanted with FLT3-ITD induced MPN lacking HIF-1alpha succumbed to their disease (average survival of 35 weeks after transplant), while animals transplanted with MPN with wild-type HIF-1alpha suffered from MPN but did not die in the observation period of 60 weeks. These findings are in contrary to what has been previously described for the role of HIF-1alpha in leukemia initiating cells and lead us to propose that HIF-1alpha could act as a tumor suppressor gene, inhibiting proliferation in myeloid malignancies. Our results provide evidence that targeting HIF-1alpha can lead to disease progression of MPN while not affecting self-renewal of LICs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ing4-Deficiency Enhances Hematopoietic Stem Cell Quiescence and Confers Resistance to Inflammatory Stress

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1095-1095
Author(s):  
Zanshé Thompson ◽  
Georgina A Anderson ◽  
Seth Gabriel ◽  
Melanie Rodriguez ◽  
Vera Binder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Loss Of Function ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

scholarly journals CRISPR genome editing of murine hematopoietic stem cells to create Npm1-Alk causes ALK+ lymphoma after transplantation

2019 ◽  
Vol 3 (12) ◽  
pp. 1788-1794 ◽  
Author(s):  
Soumya Sundara Rajan ◽  
Lingxiao Li ◽  
Mercedes F. Kweh ◽  
Kranthi Kunkalla ◽  
Amit Dipak Amin ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Large Cell ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
T Cell Lymphomas ◽  
Long Latency ◽  
Key Points ◽  
Genomic Editing

Key Points CRISPR/Cas9 genomic editing of wild-type hematopoietic stem cells generates Npm1-Alk, leading to ALK+ large-cell lymphomas in recipients. CD30+ postthymic T-cell lymphomas are polyclonal but transplantable to secondary recipients with long latency.

Erythrocyte Cytoskeletal Defects Induced in Mice by Deletion of Rac GTPases.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1573-1573 ◽  
Author(s):  
Theodosia A. Kalfa ◽  
Suvarnamala Pushkaran ◽  
James F. Johnson ◽  
Qian Wei ◽  
David A. Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Confocal Microscopy ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Erythrocyte Ghosts ◽  
Wild Type ◽  
Rhodamine Phalloidin ◽  
Hematopoietic Stem ◽  
Post Induction ◽  
Erythrocyte Cytoskeleton

Abstract The small Rho GTPases Rac1 and Rac2 have been implicated in regulating actin structures in a variety of cells, including hematopoietic stem cells and leucocytes. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. We explored the possible role of Rac1 and Rac2 signaling molecules in the dynamic assembly of actin in the red blood cells (RBC), and thus in the regulation of morphology and function of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases have been shown to have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in hematopoietic stem cells (Gu et al. Science, 2003); we focused our study on the erythrocyte phenotype of Rac2−/− and Rac1−/−;Rac2−/− mice. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background. Deletion of Rac1 after treatment with PolyI:PolyC to induce Cre recombinase was confirmed in bone marrow cells using DNA PCR and in erythrocytes by immunoblot. Since the erythrocytes consist a population of variable age, the optimal time of the maximum Rac1 deletion in erythrocytes was determined to be three to five weeks post induction. During this period, Rac1 protein in erythrocytes was decreased by 50–80% as determined by immunoblot densitometry. Rac2−/− and wild-type mice were subjected to the same treatment to control for any effects of PolyI:PolyC independent of the Rac1 deletion. Blood samples were obtained weekly after the completion of induction and the hematologic phenotype was studied by evaluation of complete blood counts, RBC indices, and reticulocyte counts. Erythrocyte morphology was examined on Wright-Giemsa smears of peripheral blood. Intact erythrocytes and erythrocyte ghosts were stained for actin with rhodamine-phalloidin and studied by confocal microscopy. The Rac2−/− mice appeared to have a rather mild erythrocyte phenotype with no significant anemia or reticulocytosis, although they did demonstrate a mild poikilocytosis and anisocytosis at baseline. The Rac1−/−;Rac2−/− mice developed a microcytic anemia with a hemoglobin drop of up to 30% in comparison to the baseline and to the wild-type hemoglobin values, with the nadir noted at three weeks post induction. The percentage of reticulocytes increased up to threefold in comparison to the control group. The mean corpuscular volume decreased up to 20% from the baseline in the Rac1−/−;Rac2−/− mice, and remained decreased up to six weeks post induction with an elevated red blood cell distribution width. Significant anisocytosis and poikilocytosis were observed with fragmented erythrocytes in the peripheral blood smear. Filamentous actin in the RBC cytoskeleton stained with rhodamine-phalloidin appeared to have a uniform distribution in intact and ghost erythrocytes under confocal microscopy. However, Rac1−/−;Rac2−/− erythrocytes demonstrated punctuate lesions on the cell surface while Rac1−/−;Rac2−/− erythrocyte ghosts appeared to collapse into irregular shapes. These data suggest that deficiency of Rac1 and Rac2 GTPases in mice cause a microcytic hemolytic anemia with poikilocytosis and red cell fragmentation indicating a possible dynamic regulation of the erythrocyte cytoskeleton organization by these signaling molecules.

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