Anaemia is frequently diagnosed in patients with cancer, yet it is difficult to identify a single cause due to its multifactorial aetiology. We conducted a systematic literature review (1996-2003) to produce evidence-based guidelines on the use of erythropoietic proteins in anaemic patients with cancer (see ). Level I evidence exists for a positive impact of erythropoietic proteins on haemoglobin (Hb) levels when administered to patients with chemotherapy-induced anaemia or anaemia of chronic disease, when used to prevent cancer anaemia, in patients undergoing cancer surgery and following allogeneic bone marrow transplantation. The Hb level at which erythropoietic protein therapy should be initiated is difficult to determine as it varied between studies; a large number of Level I studies in patients with chemotherapy-induced anaemia or anaemia of chronic disease enrolled patients with a Hb concentration </=105 g/L, but none compared the effect of different baseline Hb levels on the response to treatment. Similarly, several studies defined the target Hb concentration as 120-130 g/L following treatment with erythropoietic proteins, but none specifically addressed the correlation between target Hb level and clinical benefit in a randomised fashion. Level I evidence shows that red blood cell (RBC) transfusion requirements are significantly reduced with erythropoietic protein therapy in patients with chemotherapy-induced anaemia or when used to prevent cancer anaemia (approximately 20% reduction compared with controls). We found indirect Level I and III evidence that patients with chemotherapy-induced anaemia or anaemia of chronic disease initially classified as non-responders to standard doses proceed to respond to treatment following a dose increase (absolute increases in response rate ranged from 8% to 18%). However, none of these studies examined the effect on response rates of a longer treatment period at the lower dose, or performed a randomised comparison of a dose increase versus an unchanged dose. There is Level I evidence to show that quality-of-life (QOL) is significantly improved in patients with chemotherapy-induced anaemia and in those with anaemia of chronic disease, particularly in patients achieving a Hb response to erythropoietic protein therapy. There are insufficient data to determine the effect on survival following treatment with erythropoietic proteins in conjunction with chemotherapy or radiotherapy. There is Level I evidence that dosing of erythropoietic proteins less frequently than three times per week (TIW) is efficacious when used to treat chemotherapy-induced anaemia or prevent cancer anaemia. There is Level III evidence that initial doses of erythropoietic proteins considered to be higher than current standard practice produce higher haematological responses in patients with chemotherapy-induced anaemia or anaemia of chronic disease. Level I evidence demonstrates that several baseline patient parameters (e.g., low endogenous erythropoietin [<em>EPO</em>] concentration, age <60 years, Hb concentration>>/=90 g/L) impact upon the response to erythropoietic proteins when used to treat chemotherapy-induced anaemia or prevent cancer anaemia. Evidence indicates that endogenous <em>EPO</em> concentration impacts on response in patients with lymphoproliferative malignancies, but is not a valid parameter in patients with solid tumours. There is Level I evidence that fixed doses of erythropoietic proteins can be used at the start of therapy to treat patients with chemotherapy-induced anaemia, but maintenance doses should be titrated individually. There is no evidence that pure red cell aplasia (PRCA) occurs following treatment with erythropoietic proteins in patients with chemotherapy-induced anaemia or when used prophylactically in patients with cancer. There is Level I evidence that the risk of thromboembolic events and hypertension are slightly elevated in patients with chemotherapy-induced anaemia receiving erythropoietic proteins. Level I evidence supports the effectiveness of erythropoietic proteins to prevenroteins to prevent anaemia in non-anaemic cancer patients receiving chemotherapy or radiotherapy or in those undergoing cancer surgery. However, these are non-licensed indications and we do not currently recommend the prophylactic use of erythropoietic proteins to prevent anaemia in patients who have normal Hb values at the start of treatment. Additional trials are warranted, especially on the issues of iron replacement and cost-effectiveness of erythropoietic protein therapy, as well as on tumour response/progression and survival.

Doxorubicin (DOX) is an effective antineoplastic agent whose use has been limited by its cardiotoxic side effects. Recent studies have established that erythropoietin (EPO), a cytokine essential for red blood cell production, protects against ischemic injury in the heart and other organs. The purpose of this study was to assess whether EPO protects the heart against cardiotoxicity induced by DOX. We found that DOX-induced apoptosis and impaired heart function in mice were largely prevented by EPO administration. To investigate the mechanism of protection by EPO, cultured neonatal mouse ventricular myocytes were treated with EPO at therapeutic levels (i.e., 1 U/ml), before application of DOX (0.1-1.0 microM). EPO protected against DOX-induced cardiomyocyte death (by approximately 50%) and apoptosis assessed by annexin-V labeling, DNA fragmentation, and caspase-3 activity. DOX-mediated increases in reactive oxygen species, which trigger cardiotoxicity, were also reversed by preconditioning with EPO. These functional effects of EPO correlated with increased Akt/protein kinase B ( approximately 2-fold) and glycogen synthase kinase 3 (GSK-3; approximately 1.3-fold) phosphorylations, suggesting protection by EPO was mediated by phosphatidylinositol 3-kinase activation. Indeed, preventing Akt and GSK-3beta phosphorylations by phosphatidylinositol 3-kinase (PI3K) inhibition abolished protection by EPO against cardiomyocyte loss, apoptosis, and oxidative stress. Thus, pretreatment with therapeutic levels of EPO can protect the myocardium against DOX-induced impaired heart function and cardiomyocyte apoptosis by activating PI3K-Akt cell survival pathways.

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.

This phase 3 prospective randomized trial evaluated the efficacy and long-term safety of erythropoietin (EPO) with or without granulocyte colony-stimulating factor plus supportive care (SC; n = 53) versus SC alone (n = 57) for the treatment of anemic patients with lower-risk myelodysplastic syndromes. The response rates in the EPO versus SC alone arms were 36% versus 9.6%, respectively, at the initial treatment step, 47% in the EPO arm, including subsequent steps. Responding patients had significantly lower serum EPO levels (45% vs 5% responses for levels < 200 mU/mL vs>> or = 200 mU/mL) and improvement in multiple quality-of-life domains. With prolonged follow-up (median, 5.8 years), no differences were found in overall survival of patients in the EPO versus SC arms (median, 3.1 vs 2.6 years) or in the incidence of transformation to acute myeloid leukemia (7.5% and 10.5% patients, respectively). Increased survival was demonstrated for erythroid responders versus nonresponders (median, 5.5 vs 2.3 years). Flow cytometric analysis showed that the percentage of P-glycoprotein(+) CD34(+) marrow blasts was positively correlated with longer overall survival. In comparison with SC alone, patients receiving EPO with or without granulocyte colony-stimulating factor plus SC had improved erythroid responses, similar survival, and incidence of acute myeloid leukemia transformation.

OBJECTIVE

This efficacy study was designed to investigate traumatic brain injury (TBI) in rats treated with delayed erythropoietin (EPO) administered in a single dose compared with a triple dose.

METHODS

Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (6 animals); 2) TBI/saline group (6 animals); 3) TBI/EPOx1 group (6 animals); and 4) TBI/EPOx3 group (7 animals). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Erythropoietin (5000 U/kg) or saline was administered intraperitoneally on Day 1 (EPOx1 group) or on Days 1, 2, and 3 (EPOx3 group) postinjury. Neurological function was assessed using a modified neurological severity score, foot-fault, and Morris water maze tests. Animals were killed 35 days after injury and brain sections were stained for immunohistochemistry.

RESULTS

Compared with the saline treatment, EPO treatment in both the EPOx1 and EPOx3 groups significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved neurological functional outcome. The EPOx3 group exhibited significantly improved functional and histological outcomes compared with the EPOx1 group.

CONCLUSIONS

These data demonstrate that delayed posttraumatic administration of EPO significantly improved histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment produced better histological and functional outcomes in rats, although a single dose provided substantial benefits compared with saline treatment.

BACKGROUND

Beneficial effects of short-term erythropoietin (EPO) therapy have been demonstrated in several animal models of acute neurologic injury, including experimental autoimmune encephalomyelitis (EAE)--the animal model of multiple sclerosis. We have found that EPO treatment substantially reduces the acute clinical paralysis seen in EAE mice and this improvement is accompanied by a large reduction in the mononuclear cell infiltration and downregulation of glial MHC class II expression within the inflamed CNS. Other reports have recently indicated that peripherally generated anti-inflammatory CD4(+)Foxp3(+) regulatory T cells (Tregs) and the IL17-producing CD4+ T helper cell (Th17) subpopulations play key antagonistic roles in EAE pathogenesis. However, no information regarding the effects of EPO therapy on the behavior of the general mononuclear-lymphocyte population, Tregs or Th17 cells in EAE has emerged.

RESULTS

We first determined in vivo that EPO therapy markedly suppressed MOG specific T cell proliferation and sharply reduced the number of reactive dendritic cells (CD11c positive) in EAE lymph nodes during both inductive and later symptomatic phases of MOG(35-55) induced EAE. We then determined the effect in vivo of EPO on numbers of peripheral Treg cells and Th17 cells. We found that EPO treatment modulated immune balance in both the periphery and the inflamed spinal cord by promoting a large expansion in Treg cells, inhibiting Th17 polarization and abrogating proliferation of the antigen presenting dendritic cell population. Finally we utilized tissue culture assays to show that exposure to EPO in vitro similarly downregulated MOG-specific T cell proliferation and also greatly suppressed T cell production of pro-inflammatory cytokines.

CONCLUSIONS

Taken together, our findings reveal an important new locus whereby EPO induces substantial long-term tissue protection in the host through signaling to several critical subsets of immune cells that reside in the peripheral lymphatic system.

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.

Cobalt metabolism and toxicology are summarized. The biological functions of cobalt are updated in the light of recent understanding of cobalt interference with the sensing in almost all animal cells of oxygen deficiency (hypoxia). Cobalt (Co(2+)) stabilizes the transcriptional activator hypoxia-inducible factor (HIF) and thus mimics hypoxia and stimulates erythropoietin (Epo) production, but probably also by the same mechanism induces a coordinated up-regulation of a number of adaptive responses to hypoxia, many with potential carcinogenic effects. This means on the other hand that cobalt (Co(2+)) also may have beneficial effects under conditions of tissue hypoxia, and possibly can represent an alternative to hypoxic preconditioning. Cobalt is acutely toxic in larger doses, and in mammalian in vitro test systems cobalt ions and cobalt metal are cytotoxic and induce apoptosis and at higher concentrations necrosis with inflammatory response. Cobalt metal and salts are also genotoxic, mainly caused by oxidative DNA damage by reactive oxygen species, perhaps combined with inhibition of DNA repair. Of note, the evidence for carcinogenicity of cobalt metal and cobalt sulfate is considered sufficient in experimental animals, but is as yet considered inadequate in humans. Interestingly, some of the toxic effects of cobalt (Co(2+)) have recently been proposed to be due to putative inhibition of Ca(2+) entry and Ca(2+)-signaling and competition with Ca(2+) for intracellular Ca(2+)-binding proteins. The tissue partitioning of cobalt (Co(2+)) and its time-dependence after administration of a single dose have been studied in man, but mainly in laboratory animals. Cobalt is accumulated primarily in liver, kidney, pancreas, and heart, with the relative content in skeleton and skeletal muscle increasing with time after cobalt administration. In man the renal excretion is initially rapid but decreasing over the first days, followed by a second, slow phase lasting several weeks, and with a significant long-term retention in tissues for several years. In serum cobalt (Co(2+)) binds to albumin, and the concentration of free, ionized Co(2+) is estimated at 5-12% of the total cobalt concentration. In human red cells the membrane transport pathway for cobalt (Co(2+)) uptake appears to be shared with calcium (Ca(2+)), but with the uptake being essentially irreversible as cobalt is effectively bound in the cytosol and is not itself extruded by the Ca-pump. It is tempting to speculate that this could perhaps also be the case in other animal cells. If this were actually the case, the tissue partitioning and biokinetics of cobalt in cells and tissues would be closely related to the uptake of calcium, with cobalt partitioning primarily into tissues with a high calcium turn-over, and with cobalt accumulation and retention in tissues with a slow turn-over of the cells. The occupational cobalt exposure, e.g. in cobalt processing plants and hard-metal industry is well known and has probably been somewhat reduced in more recent years due to improved work place hygiene. Of note, however, adverse reactions to heart and lung have recently been demonstrated following cobalt exposure near or slightly under the current occupational exposure limit. Over the last decades the use of cobalt-chromium hard-metal alloys in orthopedic joint replacements, in particular in metal-on-metal bearings in hip joint arthroplasty, has created an entirely new source of internal cobalt exposure. Corrosion and wear produce soluble metal ions and metal debris in the form of huge numbers of wear particles in nanometric size, with systemic dissemination through lymph and systemic vascular system. This may cause adverse local reactions in peri-prosthetic soft-tissues, and in addition systemic toxicity. Of note, the metal nanoparticles have been demonstrated to be clearly more toxic than larger, micrometer-sized particles, and this has made the concept of nanotoxicology a crucial, new discipline. As another new potential source of cobalt exposure, suspicion has been raised that cobalt salts may be misused by athletes as an attractive alternative to Epo doping for enhancing aerobic performance. The cobalt toxicity in vitro seems to reside mainly with ionized cobalt. It is tempting to speculate that ionized cobalt is also the primary toxic form for systemic toxicity in vivo. Under this assumption, the relevant parameter for risk assessment would be the time-averaged value for systemic cobalt ion exposure that from a theoretical point of view might be obtained by measuring the cobalt content in red cells, since their cobalt uptake reflects uptake only of free ionized cobalt (Co(2+)), and since the uptake during their 120 days life span is practically irreversible. This clearly calls for future clinical studies in exposed individuals with a systematic comparison of concurrent measurements of cobalt concentration in red cells and in serum.

OBJECTIVE

Cardioprotective effects of erythropoietin (EPO) have been shown in experimental and smaller clinical studies. We performed a prospective, multicentre, randomized trial to assess the effects of a single high dose of EPO after primary coronary intervention (PCI) for an ST-elevation myocardial infarction (STEMI). Methods and results Patients with a successful PCI for a first STEMI were randomized to receive either standard medical care alone, or in combination with a single bolus with 60,000 IU i.v. of epoetin alfa within 3 h after PCI. Primary endpoint was left ventricular ejection fraction (LVEF) after 6 weeks, assessed by planar radionuclide ventriculography. Pre-specified secondary endpoints included enzymatic infarct size and major adverse cardiovascular events. A total of 529 patients were enrolled (EPO n = 263, control n = 266). At baseline (before EPO administration), groups were well-matched for all relevant characteristics. After a mean of 6.5 (± 2.0) weeks, LVEF was 0.53 (± 0.10) in the EPO group and 0.52 (± 0.11) in the control group (P = 0.41). Median area under the curve (inter-quartile range) after 72 h for creatinine kinase was 50 136 (28 212-76 664)U/L per 72 h in the EPO group and 53 510 (33 973-90 486)U/L per 72 h in the control group (P = 0.058). More major adverse cardiac events occurred in the control than in the EPO group (19 vs. 8; P = 0.032). Conclusion A single high dose of EPO after a successful PCI for a STEMI did not improve LVEF after 6 weeks. However, the use of EPO was related to less major adverse cardiovascular events and a favourable clinical safety profile.

BACKGROUND

NCT00449488; http://www.clinicaltrials.gov/ct2/show/NCT00449488?term=voors&rank=2.

OBJECTIVE

Alteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here, we examine the hypothesis that ER stress in diabetic hearts impairs phospho-glycogen synthase kinase (GSK)-3beta-mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.

METHODS

A rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with tauroursodeoxycholic acid (TUDCA) (100 mg . kg(-1) . day(-1) for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.

RESULTS

Levels of ER chaperones (GRP78 and GRP94) in the myocardium and level of nonphoshopho-GSK-3beta in the mitochondria were significantly higher in OLETF than in LETO rats. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3beta in OLETF rats. Administration of erythropoietin (EPO) induced phosphorylation of Akt and GSK-3beta and reduced infarct size (% risk area) from 47.4 +/- 5.2% to 23.9 +/- 3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3beta nor infarct size limitation was induced by EPO in OLETF rats. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF rats than in those from EPO-treated LETO rats. TUDCA restored responses of GSK-3beta, mPTP opening threshold, and infarct size to EPO receptor activation in OLETF rats. There was a significant correlation between mPTP opening threshold and phospho-GSK-3beta-to-total GSK-3beta ratio in the mitochondrial fraction.

CONCLUSIONS

Disruption of protective signals leading to GSK-3beta phosphorylation and increase in mitochondrial GSK-3beta are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.

Erythropoietin (EPO) is a 30.4 kDa glycoprotein produced by the kidney, and is mostly well-known for its physiological function in regulating red blood cell production in the bone marrow. Accumulating evidence, however, suggests that EPO has additional organ protective effects, which may be useful in the prevention or treatment of acute kidney injury. These protective mechanisms are multifactorial in nature and include inhibition of apoptotic cell death, stimulation of cellular regeneration, inhibition of deleterious pathways, and promotion of recovery.In this article, we review the physiology of EPO, assess previous work that supports the role of EPO as a general tissue protective agent, and explain the mechanisms by which it may achieve this tissue protective effect. We then focus on experimental and clinical data that suggest that EPO has a kidney protective effect.

OBJECTIVE

Critically ill patients often are anemic, which may impair oxygen delivery. Transfusion of red cells and supplementation with vitamins or iron are the usual therapeutic strategies, whereas only sporadic data are available on the use of epoetin alfa in these patients. We investigated endogenous erythropoietin (EPO) production and the response to epoetin alfa in anemic intensive care unit (ICU) patients.

METHODS

Randomized, open trial.

METHODS

Multidisciplinary ICU in a single secondary care center.

METHODS

Thirty-six critically ill patients admitted to the ICU who became anemic (hemoglobin concentration, <11.2 g/dL or <12.1 g/dL in case of cardiac disease) were randomized to one of three study groups.

METHODS

All patients received folic acid (1 mg) daily. The control group received no additional therapy, the iron group received 20 mg of iron saccharate intravenously (iv) daily for 14 days. The EPO group received iv iron and epoetin alfa (300 IU/kg) subcutaneously on days 1, 3, 5, 7, and 9.

RESULTS

Blood and reticulocyte counts were measured daily for 22 days. Serum EPO, C-reactive protein, serum transferrin receptor, and iron variables were measured on days 0, 2, 6, 10, and 21. Blood loss and red cell transfusions were recorded. Serum EPO concentrations were inappropriately low for the degree of anemia at baseline, with no difference between patients with and without renal failure. Exogenous administration of EPO increased EPO concentrations from 23+/-13 to a maximum of 166+/-98 units/L on day 10 (p < .05). Reticulocyte count increased exclusively in the EPO group from 56+/-33 x 10(9)/L to a maximum of 189+/-97 on day 13 (p < .05). Serum transferrin receptor rose only in the EPO group from 3.7+/-1.4 to 8.6+/-3.1 mg/L on day 10 (p < .05) and remained elevated on day 21, indicating an increase in erythropoiesis. Hemoglobin concentration and platelet count remained identical in the three study groups.

CONCLUSIONS

Endogenous EPO concentrations are low in critically ill patients. The bone marrow of these patients is able to respond to exogenous epoetin alfa, as shown by elevated concentrations of reticulocytes and serum transferrin receptors.

The role of nuclear protooncogenes during erythroid cell differentiation was examined by transfecting exogenous c-fos and c-myb genes into mouse erythroleukemia cells, which can be induced to differentiate either with erythropoietin (Epo) or dimethyl sulfoxide. Expression of exogenous c-myb or c-fos oncogene completely inhibited Epo-induced erythroid differentiation but only partially inhibited dimethyl sulfoxide-induced differentiation. Normally Epo-induced differentiation leads to a drastic decline of c-myb mRNA levels and an increase of c-myc transcripts in the early stage of differentiation. Cells expressing exogenous c-fos gene, however, maintained high levels of c-myb mRNA after Epo treatment. This high level of c-myb transcripts was found to be due to block of transcription shutoff (or transcriptional activation) rather than to mRNA stabilization. It is concluded that the down-regulation of endogenous c-myb gene expression is a prerequisite for commitment of Epo-induced erythroid differentiation and that expression of c-myb gene may be indirectly regulated by c-fos gene product. We also concluded that early down-regulation of c-myc gene expression is not essential for erythroid differentiation and that gene regulation of chemically induced erythroid differentiation may differ from that of Epo-induced differentiation.

Events relayed via the single transmembrane receptor for erythropoietin (Epo) are essential for the development of committed erythroid progenitor cells beyond the colony-forming unit-erythroid stage, and this clearly involves Epo's inhibition of programmed cell death (PCD). Less well resolved, however, are issues regarding the precise nature of Epo-dependent antiapoptotic mechanisms, the extent to which Epo might also promote mitogenesis and/or terminal erythroid differentiation, and the essential vs modulatory nature of certain Epo receptor cytoplasmic subdomains, signal transducing factors, and downstream pathways. Accordingly, this review focuses on the following aspects of Epo signal transduction: (1) Epo receptor/Jak2 activation mechanisms; (2) the critical vs dispensable nature of (P)Y sites and SH2 domain-encoding effectors in survival, growth, and differentiation responses; (3) primary mechanisms by which Epo inhibits PCD; (4) the integration of signals relayed by coexpressed and possibly directly interacting cytokine receptors; and (5) predictions regarding effector function which are provided by the association of certain primary and familial polycythemias with mutated human Epo receptor forms.

Microtubule-targeting agents such as the taxanes are highly active against breast cancer and have become a cornerstone in the treatment of patients with early and advanced breast cancer. The natural epothilones and their analogs are a novel class of microtubule-stabilizing agents that bind tubulin and result in apoptotic cell death. Among this family of compounds, patupilone, ixabepilone, BMS-310705, ZK-EPO, and KOS-862 are in clinical development. Extensive preclinical studies have shown that epothilones are working through partially nonoverlapping mechanisms of action with taxanes. In the clinic, epothilones have been found in a series of phase I and phase II studies to be active even in patients who had recently progressed to taxanes. The toxicity profile of these agents consists mostly of sensory neuropathy, sometimes reversible. Neoadjuvant studies with epothilones have been conducted and a number of phase III studies in advanced breast cancer are either under way or have been recently completed. The results of these studies are eagerly awaited and it is anticipated that epothilones may become an important treatment option in patients with breast cancer.

It is well known that neonatal hypoxic-ischemic brain injury leads to mental retardation and deficits in cognitive abilities such as learning and memory in human beings. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. However, the effect of Epo on cognitive abilities in the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of Epo on learning-memory, behavior and neurodegeneration induced by hypoxia-ischemia. Seven days old Wistar Albino rat pups have been used in the study (n = 28). Experimental groups in the study were: (1) saline-treated hypoxia-ischemia group, (2) Epo-treated (i.p., 1000 U/kg) hypoxia-ischemia group, (3) sham-operated group, (4) control group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was induced for 2.5 h. Epo was administered as a single dose immediately after the hypoxia period. When pups were 22 days old, learning experiments were performed using Morris water maze. On the 20th week, when brain development is accepted to be complete, learning experiments were repeated. Rats were then perfused and brains removed for macroscopic and microscopic evaluation. Epo treatment immediately after hypoxic-ischemic insult significantly improved long-term neurobehavioral achievements when tested during the subsequent phase of brain maturation and even into adulthood. Histopathological evaluation demonstrated that Epo also significantly diminished brain injury and spared hippocampal CA1 neurons. In conclusion, Epo administrated as a single dose immediately after neonatal hypoxic-ischemic insult provides benefit over a prolonged period in the still developing rat brain. Since the wide use of Epo in premature newborns, this agent may be potentially beneficial in treating asphyxial brain damage in the perinatal period.

The transforming capabilities of FVA, RLV and FVP have been examined using an in vitro transformation assay. Treatment of bone marrow cells with FVP in vitro led to the formation of hemoglobinized erythroid bursts even when these cells were cultured in methylcellulose for 5 days without added erythropoietin (Epo). A variety of FVA and RLV preparations also produced erythroid bursts without Epo but these bursts contained significantly less hemoglobin than those induced by FVP. When very low levels of Epo were added to cultures of FVA- and RLV-infected cells, the bursts were hemoglobinized, that is, similar to FVP-induced bursts. The burst-inducing agent in FVA preparations was shown to be a virus and not Epo. Spleen focus-forming virus (SFFV) pseudotypes, derived from FVA or FVP, also produced erythroid bursts in vitro, whereas four helper murine leukemia viruses did not. These studies indicated that the SFFV component was essential for erythroid burst transformation and specified the degree of hemoglobinization in the bursts formed.

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP), which was engineered to contain five N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximately 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

One of the principal functions of erythropoietin (EPO) is to stimulate the maturation of erythroid precursors. Yet EPO has recently been shown to modulate a host of cellular signal transduction pathways in pluripotent stem cells to perform multiple functions other than erythropoiesis. The production of EPO is tightly modulated by the loss of oxygen and the hypoxia-inducible factor 1. Once generated, EPO becomes a robust stimulus which regulates endothelial cell proliferation and migration as well as erythropoiesis and vascular resistance. Further downstream in the signal transduction cascade, EPO engages diverse cellular pathways--such as those involving Janus kinase 2, signal transducers and activators of transcription (STATs), mitogen-activated protein kinases (MAPKs), Bcl-x(L), protein kinase B, protein kinase C, and cysteine proteases--to provide "plasticity" to vascular systems through highly conserved mechanisms. EPO also has recently been demonstrated to inhibit the induction of apoptosis through two distinct components that involve the maintenance of the integrity of genomic DNA and the preservation of cellular membrane asymmetry. Recognition of the multipotential attributes of EPO for vascular systems may further the progress of the development of therapeutic strategies to delay the onset of degenerative diseases.

Tissue hypoxia is a characteristic property of cervical cancers that makes tumors resistant to chemo- and radiation therapy. Erythropoietin (Epo) is a hypoxia-inducible stimulator of erythropoiesis. Acting via its receptor (EpoR), Epo up-regulates bcl-2 and inhibits apoptosis of erythroid cells and rescues neurons from hypoxic damage. In addition to human papillomavirus infection, increased bcl-2 expression and decreased apoptosis are thought to play a role in the progression of cervical neoplasia. Using reverse transcriptase-polymerase chain reaction and Western blotting we showed that HeLa and SiHa cervical carcinoma cells and human cervical carcinomas express EpoR, and that hypoxia enhances EpoR expression. Exogenous Epo stimulated tyrosine phosphorylation and inhibited the cytotoxic effect of cisplatin in HeLa cervical carcinoma cells. Using immunohistochemistry, we examined the expression of Epo, EpoR, p16, hypoxia-inducible factor (HIF)-1alpha, and bcl-2 in benign and dysplastic cervical squamous epithelia and invasive squamous cell carcinomas (ISCCs). EpoR expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse EpoR expression was found in all ISCCs. Expression of Epo and HIF-1alpha was increased in dysplasias compared to benign epithelia. Focal Epo and HIF-1alpha expression was seen near necrotic areas in ISCCs, and showed correlation in their spatial distribution. Significant correlation was found between expression of EpoR, and p16 and bcl-2 in benign and dysplastic squamous epithelia. Our results suggest that increased expression of Epo and EpoR may play a significant role in cervical carcinogenesis and tumor progression. Hypoxia-inducible Epo signaling may play a significant role in the aggressive behavior and treatment resistance of hypoxic cervical cancers.

BACKGROUND

The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing.

RESULTS

In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression.

CONCLUSIONS

Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.