ALBERT

Description: Abstract 3186 Identification of new key players in erythropoiesis can lead to a better understanding of the etiology of anemia of unknown origin. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. Crisis episodes are characterized by severe anemia and thrombocytopenia while, remarkably, the phenotype reverts to normal in the remission phase. Remissions are transient, however, and 94% of scat/scat mice die by P30 during a second crisis episode. We recently demonstrated a mechanism contributing to crisis episodes. The G125V mutation in Rasa3 is a loss of function mutation causing the protein to be mislocalized to the cytosol in scat reticulocytes. This results in loss of GAP activity and, as a consequence, increased levels of active GTP-bound Ras and a severe block in erythroid differentiation. Morpholino knockdowns of rasa3 in zebrafish result in profound anemia, confirming a conserved and non-redundant role for Rasa3 in vertebrate erythropoiesis. Here, we report that the cell cycle is affected in scat erythroid progenitors and extend studies to human primary erythroid cells. Using propidium iodine and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, × ± SD p

Description: In order to study 45Ca distribution within erythrocytes, a method was devised that had minimal deleterious effects on the treated erythrocytes. It was observed that newly introduced 45Ca was predominantly recoverable from the cytosol and exchanged relatively slowly with membrane-associated Ca. Younger erythrocytes appeared to have relatively more 45Ca in membrane-associated sites. Erythrocytes from patients with sickle cell anemia had significantly more 45Ca in membrane-associated sites than did normal controls or patients with reticuloctosis due to a variety of disorders. There are theoretical reasons for considering the possibility that the distribution of 45Ca between cytosol and membrane-associated sites could modulate some of the properties of the erythrocyte membrane.

Description: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2237 Background: Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes (IBMFS), characterized by erythroid hypoplasia, congenital anomalies and a cancer predisposition. DBA is caused by ribosomal protein haploinsufficiency, which somehow triggers apoptosis of erythroid precursors, possibly through activation of p53. Some DBA patients show a response to steroid treatment, while others remain transfusion-dependent. While the mechanism of action of steroids in DBA is unclear, recent work has suggested that p53 may antagonize glucocorticoid-induced proliferation of normal erythroid progenitors1. Objective: Our goal was to create a murine embryonic stem (ES) cell model of DBA with a mutation in Rps19 to study the pathophysiology of DBA and to test glucocorticoid responsiveness. Methods: The Rps19-mutated murine embryonic stem cell line, S17-10H1 was created using a gene trap strategy. The ES cells were induced to undergo primary differentiation into embryoid bodies (EBs). Day 9–11 EBs, representing definitive hematopoiesis, were re-plated with hematopoietic cytokines (stem cell factor, interleukin (IL)-3, IL-6 and erythropoietin) in methylcellulose, and secondary differentiated colonies were scored on day 10. S17-10H1 cells were transfected by electroporation with a plasmid vector expressing either wild-type Rps19 cDNA or an empty vector control. Protein and mRNA levels of the tumor suppressor p53 were measured at the ES cell stage. Secondary differentiated hematopoietic colonies were grown with and without glucocorticoids (either dexamethasone at 1nM, 10nM, 100nM, 1μM or hydrocortisone at 50nM, 2.5μM concentration). Results: Western blot analyses confirmed S17-10H1 Rps19-haploinsufficiency. The mutant cell line had reduced EB formation (hematopoietic and non-hematopoietic) following primary differentiation. Significant defects in both erythroid (BFU-E) and myeloid (CFU-GM) formation were found following secondary hematopoietic differentiation of day 9–11 EBs. These defects were specific to Rps19 haploinsufficiency since all defects were rescued by stable transfection of the mutant cell lines with an Rps19-expressing correction vector. However, glucocorticoid treatment was unable to rescue defects in secondary differentiation (hematopoietic colony formation). Finally, we also examined baseline levels of the tumor suppressor p53, which may be induced following abortive ribosome assembly resulting from ribosomal protein haploinsufficiency. Although there was no difference in transcription levels of p53 mRNA between mutant and control cells as determined by quantitative PCR, baseline levels of p53 protein were significantly increased in the mutant ES cells as compared to controls. Conclusions: (1) In our model system, Rps19 mutation was associated with a relatively early defect in hematopoietic progenitors, since both erythroid and myeloid (or mixed) colony formation was diminished. (2) Hematopoietic defects in our murine ES cell model of DBA were rescued by Rps19 gene transfer but not by glucocorticoid therapy. (3) We found significant accumulation of baseline p53 protein (but not mRNA) levels in mutant ES cells, suggesting that Rps19 haploinsufficiency leads to inhibition of the p53-degradation pathway. (4) Taken together, these results suggest that p53 may antagonize glucocorticoid-induced proliferation in our Rps19-mutant ES cell model. Experiments to test this hypothesis are in progress. Reference: Ganguli G, Back J, Sengupta S, Wasylyk B. The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors. EMBO Rep. 2002;3:569-574. Disclosures: No relevant conflicts of interest to declare.

Description: Previous reports have suggested that platelets from patients with Bernard-Soulier's syndrome (BSS) are not giant cells. Rather, they are normal-sized in suspension, but spread out on glass slides more readily than control cells, yielding the impression of being giant. The present study has used cell sizing techniques, electron microscopy, and micropipette aspiration to evaluate platelets from three patients with BSS. Cell sizing techniques revealed that BSS platelets were considerably larger than normal. The increased size was confirmed in electron microscopic studies of BSS platelets fixed in suspension. However, the BSS platelets did not contain increased amounts of internalized surface membrane considered to be the source of membrane necessary for excessive spreading. A possible explanation for increased spreading of BSS platelets was found in studies of their resistance to deformation in micropipettes. BSS platelets were much less resistant to deformation than normal cells or other abnormal platelets when aspirated under the same negative pressure. Their unusual deformability may explain the tendency of BSS platelets to spread more readily than normal cells on glass slides.

Description: In order to study 45Ca distribution within erythrocytes, a method was devised that had minimal deleterious effects on the treated erythrocytes. It was observed that newly introduced 45Ca was predominantly recoverable from the cytosol and exchanged relatively slowly with membrane-associated Ca. Younger erythrocytes appeared to have relatively more 45Ca in membrane-associated sites. Erythrocytes from patients with sickle cell anemia had significantly more 45Ca in membrane-associated sites than did normal controls or patients with reticuloctosis due to a variety of disorders. There are theoretical reasons for considering the possibility that the distribution of 45Ca between cytosol and membrane-associated sites could modulate some of the properties of the erythrocyte membrane.

Description: In order to study 45Ca movements within erythrocytes, a method was devised that had minimal deleterious effect on the treated erythrocytes. Agents that induce endocytosis in intact erythrocytes (primaquine, vinblastine, and chlorpromazine) caused a prompt movement of 45Ca from cytosol to membrane-associated sites. This drug-induced movement of 45Ca to membrane sites was blocked by depleting erythrocytes of adenosine triphosphate (ATP) or by incubating them with known inhibitors of endocytosis, NaF of N-ethylmaleimide (NEM). It appears that endocytosis in intact human erythrocytes involves the movement and redistribution of 45Ca from cytosol to membrane-associated sites. Therefore, in the erythrocyte, as in perhaps other cells, movement of Ca from one site to another may modulate important cellular biologic functions.

Description: Abstract 1270 Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6. Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p

Description: Previous reports have suggested that platelets from patients with Bernard-Soulier's syndrome (BSS) are not giant cells. Rather, they are normal-sized in suspension, but spread out on glass slides more readily than control cells, yielding the impression of being giant. The present study has used cell sizing techniques, electron microscopy, and micropipette aspiration to evaluate platelets from three patients with BSS. Cell sizing techniques revealed that BSS platelets were considerably larger than normal. The increased size was confirmed in electron microscopic studies of BSS platelets fixed in suspension. However, the BSS platelets did not contain increased amounts of internalized surface membrane considered to be the source of membrane necessary for excessive spreading. A possible explanation for increased spreading of BSS platelets was found in studies of their resistance to deformation in micropipettes. BSS platelets were much less resistant to deformation than normal cells or other abnormal platelets when aspirated under the same negative pressure. Their unusual deformability may explain the tendency of BSS platelets to spread more readily than normal cells on glass slides.