Mark J. Koury

Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death

The gene FUS (also known as TLS (for translocated in liposarcoma) and hnRNP P2) is translocated with the gene encoding the transcription factor ERG-1 in human myeloid leukaemias. Although the functions of wild-type FUS are unknown, the protein contains an RNA-recognition motif and is a component of nuclear riboprotein complexes. FUS resembles a transcription factor in that it binds DNA, contributes a transcriptional activation domain to the FUS–ERG oncoprotein and interacts with several transcription factors in vitro. To better understand FUS function in vivo, we examined the consequences of disrupting Fus in mice. Our results indicate that Fus is essential for viability of neonatal animals, influences lymphocyte development in a non-cell-intrinsic manner, has an intrinsic role in the proliferative responses of B cells to specific mitogenic stimuli and is required for the maintenance of genomic stability. The involvement of a nuclear riboprotein in these processes in vivo indicates that Fus is important in genome maintenance.

New insights into erythropoiesis.

Commitment of hematopoietic cells to the erythroid lineage involves the actions of several transcription factors, including TAL1, LMO2, and GATA-2. The differentiation of committed erythroid progenitor cells involves other transcription factors, including NF-E2 and EKLF. Upon binding erythropoietin, the principal regulator of erythropoiesis, cell surface erythropoietin receptors dimerize and activate specific intracellular kinases, including Janus family tyrosine protein kinase 2, phosphoinositol-3 kinase, and mitogen-activated protein kinase. Important substrates of these kinases are tyrosines in the erythropoietin receptors themselves and the signal transducer and transcription activator proteins. Erythropoietin prevents erythroid cell apoptosis. Some of the apoptotic tendency of erythroid cells can be attributed to proapoptotic molecules produced by hematopoietic cells, macrophages, and stromal cells. Cell divisions accompanying terminal erythroid differentiation are finely controlled by cell cycle regulators, and disruption of these terminal divisions causes erythroid cell apoptosis. In reticulocyte maturation, regulated degradation of internal organelles involves a lipoxygenase, whereas survival requires the antiapoptotic protein Bcl-x.

Erythropoietin messenger RNA levels in developing mice and transfer of 125I-erythropoietin by the placenta.

Erythropoietin (EP) mRNA was measured in normal and anemic mice during fetal and postnatal development. Normal fetal livers at 14 d of gestation contained a low level of EP mRNA. By day 19 of gestation, no EP mRNA was detected in normal or anemic fetal livers or normal fetal kidneys, but anemic fetal kidneys had low levels of EP mRNA. Newborn through adult stage mice responded to anemia by accumulating renal and hepatic EP mRNA. However, total liver EP mRNA was considerably less than that of the kidneys. Juvenile animals, 1-4 wk old, were hyperresponsive to anemia in that they produced more EP mRNA than adults. Moreover, nonanemic juveniles had readily measured renal EP mRNA, whereas the adult level was at the lower limit of detection. Because of the very low level of fetal EP mRNA, placental transfer of EP was evaluated. When administered to the pregnant mouse, 125I-EP was transferred in significant amounts to the fetuses. These results indicate that in mice the kidney is the main organ of EP production at all stages of postnatal development and that adult kidney may also play some role in providing EP for fetal erythropoiesis via placental transfer of maternal hormone.

Ferric Citrate Controls Phosphorus and Delivers Iron in Patients on Dialysis

Patients on dialysis require phosphorus binders to prevent hyperphosphatemia and are iron deficient. We studied ferric citrate as a phosphorus binder and iron source. In this sequential, randomized trial, 441 subjects on dialysis were randomized to ferric citrate or active control in a 52-week active control period followed by a 4-week placebo control period, in which subjects on ferric citrate who completed the active control period were rerandomized to ferric citrate or placebo. The primary analysis compared the mean change in phosphorus between ferric citrate and placebo during the placebo control period. A sequential gatekeeping strategy controlled study-wise type 1 error for serum ferritin, transferrin saturation, and intravenous iron and erythropoietin-stimulating agent usage as prespecified secondary outcomes in the active control period. Ferric citrate controlled phosphorus compared with placebo, with a mean treatment difference of -2.2±0.2 mg/dl (mean±SEM) (P<0.001). Active control period phosphorus was similar between ferric citrate and active control, with comparable safety profiles. Subjects on ferric citrate achieved higher mean iron parameters (ferritin=899±488 ng/ml [mean±SD]; transferrin saturation=39%±17%) versus subjects on active control (ferritin=628±367 ng/ml [mean±SD]; transferrin saturation=30%±12%; P<0.001 for both). Subjects on ferric citrate received less intravenous elemental iron (median=12.95 mg/wk ferric citrate; 26.88 mg/wk active control; P<0.001) and less erythropoietin-stimulating agent (median epoetin-equivalent units per week: 5306 units/wk ferric citrate; 6951 units/wk active control; P=0.04). Hemoglobin levels were statistically higher on ferric citrate. Thus, ferric citrate is an efficacious and safe phosphate binder that increases iron stores and reduces intravenous iron and erythropoietin-stimulating agent use while maintaining hemoglobin.

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b561 (Cyt b561) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b561, immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b561. In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Comparative Analysis of Different Methodological Approaches to the in Vitro Study of Drug-Induced Apoptosis

Apoptosis is a dynamic process in which a characteristic morphological or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time. Asynchronous involvement of cells in apoptosis results in different proportions of apoptotic cells with blebbed membrane, broken nuclei, modified mitochondrial units or fragmented DNA coexisting in the culture at any single moment. Thus, depending on the method used, the extent of apoptosis determined in the same cell population may vary. In the present study, a microculture kinetic (MiCK) assay was used to monitor apoptosis in HL-60 cells exposed to 1, 2.5, 5, 10, and 20 micromol/L etoposide and cisplatin. Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video microscopy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, and DNA fragmentation assays were performed at multiple times of cell exposure to 10 micromol/L etoposide and 5 micromol/L cisplatin. Steep linear increases in optical density, indicating apoptosis in the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebbing in TLVM and with increased side scattering properties of apoptotic cells in flow cytometry. During a 24-hour culture period, the MiCK assay and TLVM provided multiple consecutive appraisals of nondisturbed cell microcultures at intervals of 5 and 2.5 minutes, respectively, and thus could be considered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic responses varied from 22.5 to 72% in etoposide-treated cells and from 30 to 57% in cisplatin-treated cells. With the annexin V binding assay, maximum apoptosis could always be detected 4 to 5 hours earlier than it was seen in Giemsa-stained preparations and 8 hours earlier than it was detected by measuring of DNA fragmentation. Values of the maximum extent of apoptosis varied, being the lowest with annexin V and the greatest with DNA fragmentation assays. The best correlations of both extent and timing of apoptosis were observed between the MiCK, TLVM, and morphological assays. In conclusion, both a maximum apoptotic response and the time at which it was achieved are the obligatory requirements for determining the apoptosis-inducing potency of an agent and for comparing results of studies performed in different laboratories.

Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch

A physiologically important alternative pre‐mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE16) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre‐mRNAs showed that CE16 can repress splicing of upstream introns, and that mutagenesis or replacement of CE16 can relieve this inhibition. An affinity selection assay with biotinylated CE16 RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage‐specific activation of the E16 splicing switch in concert with a dramatic and specific down‐regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre‐mRNA splicing.

Control of red cell production: the roles of programmed cell death (apoptosis) and erythropoietin

The production of red cells (RBCs) in mammals is controlled by erythropoietin (EPO). EPO is a glycoprotein hormone of molecular weight 34,000 that is produced by peritubular interstitial ce!ls in the renal cortex in response to hypoxia.’ Erythroid progenitor cells in the bone marrow develop into reticulocytes in response to EPO, but the mechanism by which EPO controls this development is unknown. Possible mechanisms of EPO action include the stimulation of mitosis, the induction of a program of terminal maturation, and the maintenance of cellular viability during a preprogrammed differentiation period. Earlier studies about the prevalence of colony-forming units-erythroid (CFU-E)2 in vivo and about the EPO-mediated growth and differentiation of CFU-E in vitro can be explained by EPO’s facilitation of the survival of these erythroid progenitor cells rather than direct stimulation of their DNA synthesis and cell division. These studies showed a) that in vivo, low EPO concentrations lead to decreased CFU-E numbers and high EPO concentrations lead to increased numbers of CFU-E, but the percentages of CFU-E in DNA synthesis are similar at all EPO levels;3 b) that CFU-E are lost after only a few hours in culture without EPO and cannot be rescued by subsequent EPO a d d i t i ~ n ; ~ ~ and c) that the numbers of CFU-E that survive and differentiate in vitro are directly related to EPO concentration, and many potential CFU-E require EPO concentrations that are higher than those found in normal More recent studies with populations of purified late-stage erythroid progenitor cells, including those of the CFU-E stage, have also indicated that these progenitor cells require EPO for survival in vitro.9*10 Thus, at least in the case of erythroid progenitor cells at the CFU-E stage of development, EPO appears to act by maintaining cellular viability. To study the mechanism of EPO action requires a population of erythroid progenitor cells that is developmentally homogeneous, responsive to EPO in vitro, and large enough to permit serial biochemical studies. Such a cell population can be obtained from spleens of mice infected with the anemia-inducing strain of Friend leukemia virus (FVA cells). FVA cells are CFU-E and the immediate progeny of CFU-E-the proerythrob1asts.I’ When cultured with physiologic concentrations of EPO, FVA cells differentiate into reticulocytes in 48 hours. When cultured without EPO, FVA cells fail to differentiate and begin to die after 16 hours.g When FVA cells die as a result of EPO deprivation, their death has characteristics of a process known as apoptosis, or programmed cell death.” Cells undergoing apoptosis show decreases in volume, condensation of nuclei, and interControl of red cell production: the roles of programmed cell

Ferric Citrate Reduces Intravenous Iron and Erythropoiesis-Stimulating Agent Use in ESRD

Ferric citrate (FC) is a phosphate binder with shown efficacy and additional effects on iron stores and use of intravenous (iv) iron and erythropoiesis-stimulating agents (ESAs). We provide detailed analyses of changes in iron/hematologic parameters and iv iron/ESA use at time points throughout the active control period of a phase 3 international randomized clinical trial. In all, 441 subjects were randomized (292 to FC and 149 to sevelamer carbonate and/or calcium acetate [active control (AC)]) and followed for 52 weeks. Subjects on FC had increased ferritin and transferrin saturation (TSAT) levels compared with subjects on AC by week 12 (change in ferritin, 114.1±29.35 ng/ml; P<0.001; change in TSAT, 8.62%±1.57%; P<0.001). Change in TSAT plateaued at this point, whereas change in ferritin increased through week 24, remaining relatively stable thereafter. Subjects on FC needed less iv iron compared with subjects on AC over 52 weeks (median [interquartile range] dose=12.9 [1.0-28.9] versus 26.8 [13.4-47.6] mg/wk; P<0.001), and the percentage of subjects not requiring iv iron was higher with FC (P<0.001). Cumulative ESA over 52 weeks was lower with FC than AC (median [interquartile range] dose=5303 [2023-9695] versus 6954 [2664-12,375] units/wk; P=0.04). Overall, 90.3% of subjects on FC and 89.3% of subjects on AC experienced adverse events. In conclusion, treatment with FC as a phosphate binder results in increased iron parameters apparent after 12 weeks and reduces iv iron and ESA use while maintaining hemoglobin over 52 weeks, with a safety profile similar to that of available binders.