Arsenic sulfide nanoformulation induces erythroid differentiation in chronic myeloid leukemia cells through degradation of BCR-ABL.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disorder due to the existence of BCR-ABL fusion protein that allows the cells to keep proliferating uncontrollably. Although tyrosine kinase inhibitors can inhibit the activity of BCR-ABL fusion protein to trigger the cells apoptosis, drug resistance or intolerance exists in part of CML patients. Arsenic sulfide in its raw form (r-As4S4) can be orally administrated and certain therapeutic effects have been found out in the treatment of hematologic malignancies through inducing cell apoptosis. METHODS: In this work, a water-dissolvable arsenic sulfide nanoformualtion (ee-As4S4) composed of As4S4 particulates with 470 nm in diameter and encapsulated by a kind of hydrophilic polymer was fabricated and applied to the CML cell line K562, K562/AO2 and primary cells from the bone marrow of CML patients. RESULTS: Results showed that instead of inhibiting the activity of BCR-ABL, ee-As4S4 induced direct degradation of BCR-ABL in K562 cells within 6 hr incubation, followed by the occurrence of erythroid differentiation in K562 after 72 hr incubation, evidenced by the significantly upregulated CD235a and benzidine staining, which was not detectable with r-As4S4. The ee-As4S4-induced erythroid differentiation was also observed in K562/AO2 cells and bone marrow mononuclear cells of CML patients. Mechanistic studies indicated that ee-As4S4 induced autophagy by downregulating the level of intracellular ROS and hypoxia-inducible factor-1α significantly, which led to the subsequent degradation of BCR-ABL. When the concentration was increased, ee-As4S4 induced much more significant apoptosis and cell cycle arrest than r-As4S4, and the cytotoxicity of the former was about 178 times of the latter. CONCLUSION: ee-As4S4 was capable of inducing significant erythroid differentiation of CML cells by inducing the direct degradation of BCR-ABL; the new effect could improve hematopoietic function of CML patients as well as inhibit the leukemic cell proliferation.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Cdk6 contributes to cytoskeletal stability in erythroid cells.

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis.

Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.

Common fragile site profiling in epithelial and erythroid cells reveals that most recurrent cancer deletions lie in fragile sites hosting large genes.

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes.

Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.

Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells.

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Autoimmune diseases caused by defects in clearing dead cells and nuclei expelled from erythroid precursors.

Apoptotic cells are recognized and subsequently engulfed by macrophages and immature dendritic cells. The engulfed dead cells are transported to the lysosomes of macrophages, and their components are degraded into amino acids and nucleotides for reuse. In mammals, macrophages also engulf nuclei expelled from erythroid precursors in the final stage of definitive erythropoiesis. Failure to swiftly engulf dead cells at the germinal centers of lymphoid organs causes systemic lupus erythematosus-type autoimmune diseases. In contrast, failure to efficiently degrade the DNA of dead cells or erythroid cell nuclei activates innate immunity, causing lethal anemia in the fetus and chronic arthritis in adults.

Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.

Survival of parvovirus B19-infected cells by cellular autophagy.

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.

Erythroid cell differentiation is characterized by nuclear matrix localization and phosphorylation of protein kinases C (PKC) alpha, delta, and zeta.

Protein kinases C (PKC) zeta expression and phosphorylation at nuclear level during dimethyl sulfoxide (DMSO)-induced differentiation in Friend erythroleukemia cells have been previously reported, suggesting a possible role of this PKC isoform in the DMSO-related signaling. In order to shed more light on this tantalizing topic, we investigated PKC intracellular and sub-cellular localization and activity during DMSO-induced erythroid differentiation. Results indicated that at least PKC alpha, zeta, and delta are strongly and temporally involved in the DMSO-induced differentiation signals since their expression and phosphorylation, though at different extents, were observed during treatments. Intriguingly, while PKC alpha and zeta associate to the nuclear matrix during the differentiation event, PKC delta appears to be residentially associated to the nuclear matrix. Furthermore, an evident downregulation of the beta-globin gene transcription (differentiation hallmark) was detected upon a progressive inhibition of these PKC isoforms by means of specific inhibitors, indicating, therefore, that PKC alpha, zeta, and delta phosphorylation play a crucial role in the control of erythroid differentiation.

Haemoglobin biosynthesis site in rabbit embryo erythroid cells.

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.