The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

It was hypothesized that, in shift workers with a history of intermittent hypoxic stress (working 10 days at > 3,600 m, then resting for 4 days at sea level) for > 5 yr, the initial erythropoietin (EPO) response and the changes in central venous pressure (CVP) are different from those in Caucasian lowlanders. We studied the kitchen personnel (n = 11) of a mine (3,600 m) and a group of Caucasian lowlanders (n = 5). Blood samples were taken, and CVP was determined several times before, during, and after a typical shift. At baseline data collection (BDC) before transition, the shift workers had EPO concentrations of 5.2 +/- 2.4 mU/ml, which increased at altitude (P < 0.01) and returned to BDC values on the recovery (day 16). The Caucasians showed the same time course. Serum transferrin receptor concentrations did not change in either group. CVP values were generally higher in the shift workers than in the Caucasians. In conclusion, the hypothesis that the initial EPO response to a hypoxic stimulus is altered in these shift workers has to be refuted. Higher hemoglobin concentrations and/or CVP values in shift workers might be responsible for the rather low EPO concentrations in shift workers at BDC.

The aim of this study was to analyze the time course of erythropoietin (EPO) during Earth-bound microgravity simulations such as bed rest, isolation and confinement (IC), head-down tilt (HDT; -6 degrees), and immersion to evaluate which factors could contribute to alterations in EPO under real microgravity conditions during and after short- (< 10 days) and long-term (> 6 mo) spaceflights. During bed rest (24h), no significant changes in EPO could be observed. Subjects confined in a diving chamber facility for 60 days showed a decrease in EPO. In the recovery period a slight increase was observed, but EPO concentrations did not reach the pre-IC control level. In the control period before HDT, subjects showed normal resting values for EPO, but on day 2 of HDT the EPO concentrations were decreased (P < 0.01). Later the EPO levels remained below the control value and were increased after HDT (P < 0.05). After immersion (24 h) increased EPO concentrations could be determined (P < 0.05). During a short-term spaceflight the astronauts showed in-flight (day 4) decreased and unchanged EPO concentrations. During a long-term spaceflight, 24 h after recovery, the cosmonaut showed slightly elevated EPO concentration, which increased markedly during the following days. It is concluded that 1) HDT (-6 degrees) causes a rapid decrease in EPO in humans, 2) IC per se leads to diminished EPO concentrations, 3) EPO regulation in humans during short- and long-term spaceflights might be different, 4) changes in central blood volume, i.e., central venous pressure, seem to be involved in the modulation of EPO production and release under simulated and real microgravity conditions, and 5) the HDT (-6 degrees) Earth-bound simulation reflects mostly the changes in EPO production and release observed under real microgravity conditions in humans.

The effect of reduced oxygen tension and the role of cellular components known to protect the cell against oxygen toxicity has been studied with respect to erythropoietic colony formation in vitro. Alphathioglycerol can be partially replaced by vitamin E and completely replaced by reduced glutathione (GSH) at physiological concentrations. Incubation of bone marrow and fetal liver early (BFU-E) and late (CFU-E) erythropoietic progenitor cells, in the presence of GSH, in an atmosphere containing 5% oxygen, 5% carbon dioxide and 90% nitrogen, as opposed to air supplemented with 5% carbon dioxide, resulted in an increase in colony numbers and response to erythropoietin (Epo). The number of colonies derived from bone marrow and fetal liver CFU-E increased by 1.2--2.8-fold with a relative Epo sensitivity increase of 3.5--4-fold. Bursts obtained from bone marrow and fetal liver BFU-E increased from 2.6- to 3.8-fold with an increased response to Epo of 2--3-fold. The effects of GSH and low oxygen tension are interpreted as causing a reduction in oxygen toxicity of the cells, thereby increasing the life span in vitro and so increasing the number of cells capable of forming colonies. The heightened response of BFU-E to Epo, analogous to the effect seen for CFU-E, implies that BFU-E may be responsive to physiological Epo concentrations at physiological oxygen tensions.

The lympho-hematopoietic colony-forming assay has been redesigned into a rapid, nonsubjective and standardized proliferation assay that can measure the effects of compounds on multiple stem and progenitor cell populations from different species simultaneously using a sensitive, high-throughput bioluminescence readout. Eleven reference compounds from the Registry of Cytotoxicity (RC) and eight other compounds, including anticancer drugs, were studied over an 8- to 9-log dose range for their effects on seven cell populations from both human and mouse bone marrow simultaneously. The cell populations studied included a primitive (HPP-SP) and mature (CFC-GEMM) stem cell, three hematopoietic (BFU-E, GM-CFC, Mk-CFC) and two lymphopoietic (T-CFC, B-CFC) populations. The results reveal a five-point prediction paradigm for lympho-hematotoxicity. Depending on how and which populations are affected, the resulting effects in the periphery can be predicted. Validation against the RC Prediction Model produces a high degree of correlation between the in vitro IC(50) values and known in vivo LD(50) values, thereby allowing preclinical dosing to be predicted. If primary human hematopoietic target tissue is used, inhibitory concentration (IC(50)/IC(75)/IC(90)) values of anticancer and other drugs can be converted into predicted clinical doses which, when compared to published chemotherapeutic dosing regimen, are very similar. When performed during early drug screening, the prediction value of the assay should help reduce time and cost, but above all, provide increase efficacy and safety for the patient.

Despite the many innovations that have occurred in cancer treatment, the age-specific mortality for most adult tumors has remained stable during the past 30 years. There have been clinically significant improvements in the outcomes of young and middle-aged patients, yet the vast majority of cancer patients are more than 50 years of age, among whom we observe few improvements in clinical outcomes. Clearly, many of today's cytotoxic agents have been shown to be effective in-vitro and in animal model systems; however, few have proved efficacious in dramatically improving survival outcomes in adult cancer. There is now increasing evidence to suggest that the administration of cytotoxic agents, at the appropriate circadian phase, can significantly increase the therapeutic index of current cancer therapies.

The identity of the cells giving rise to the hematopoietic system in the mouse embryo are unknown. The results presented here strongly suggest that hematopoietic cells are derived from a nonhematopoietic cell population that has been previously thought to give rise to the germ cells. These cells are called primordial germ cells (PGCs) and can be recognized as large cells showing blebbing and pseudopodial extrusions on their surface. They are alkaline phosphatase (AP) positive and possess a stage-specific embryonic antigen (SSEA-1) on their surface. They represent a small pool of cells in the extraembryonic mesoderm at the base of the allantois in late day-6 embryos. Primordial germ cells from 7.5- and 8.5-day visceral yolk sac and embryo proper form AP+ and SSEA-1+ colonies within 5 days when grown on an embryonic fibroblast feeder cell layer in the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and interleukin-3 (IL-3). Individual colonies taken from day-5 cultures can be shown to differentiate into erythroid lineage cells in secondary methyl cellulose culture and produce secondary and tertiary PGCs in the presence of LIF, SCF, and IL-3. Cells taken from the region of the allantois and primitive streak can form colonies on hydrophilic Teflon (DuPont, Wilmington, DE) foils precoated with collagen and fibronectin. The cells from these colonies were then shown to form cobblestone areas on irradiated adult bone marrow stromal layers, indicating that the most primitive in vitro hematopoietic stem cell, the cobblestone-area forming cell (CAFC), was present. PGC colonies were grown in methyl cellulose in the presence of LIF, SCF, and IL-3 for 5 days, and the colonies were removed and passaged 3 times on pretreated extracellular matrix hydrophilic Teflon foils. After each passage, the cells were assayed for their differentiation capacity and PGC content. After the last passage, the number of CAFCs was also determined. It was found that, under these conditions, the PGC population expanded more than 400- fold and also contained CAFCs. It is postulated that the PGC represents a totipotent stem cell population capable of producing a variety of different cell types including cells of the hematopoietic system.

Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron-deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron-deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti-thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre-treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit. Results of this study argue against the model of a simple feedback regulation of EPO via hypoxic anaemia. Our data support the hypothesis that cytokines and the erythropoietic progenitor pool are involved in the regulation of EPO production. The results illustrate that serial measurements of EPO along with therapeutic interventions are necessary to identify patients who might benefit from treatment with exogenous recombinant human EPO.