Vivien M. Hodges

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA–mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread. Mol Cancer Res; 8(4); 615–26. ©2010 AACR.

Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli

Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined. Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EPO was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive. The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis. Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins. Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.

Characterization and localization of expression of an erythropoietin-induced gene, ERIC-1/TACC3, identified in erythroid precursor cells

Gene expression profiles during erythropoietin (Epo)‐induced differentiation of erythroid progenitor cells derived from the Friend virus anaemia (FVA) and phenylhydrazine (PHZ) murine models have been examined using differential display polymerase chain reaction (PCR). Ten cDNA fragments upregulated by Epo were isolated. The ribonuclease protection assay confirmed differential expression between Epo‐stimulated and Epo‐deprived cells for one of these, provisionally named ERIC‐1. Sequencing of the full‐length cDNA predicted a protein of 558 amino acids, 17 amino acids longer than mTACC3, the third member of a novel family of proteins that contain a coiled‐coil domain. The human homologue, cloned using rapid amplification of cDNA ends (RACE)‐PCR, encodes a larger protein of 838 amino acids that is identical to hTACC3. In addition to erythroid precursor cells, ERIC‐1/TACC3 is expressed at high levels in the testes, at moderate levels in the thymus and peripheral leucocytes, and at lower levels in the spleen and intestinal tissue. Immunohistochemical analysis using an antibody to a GST fusion product of the C‐terminus of hERIC‐1/TACC3 revealed that it is localized to Sertoli cells in the human testes. Confocal microscopy demonstrated hERIC‐1/TACC3 protein concentrated in the perinuclear vesicles of dermal microvascular endothelial cells. Although ERIC‐1/TACC3 is expressed in a wide range of tissues, its upregulation by Epo in erythroid progenitors implies that it has a role in terminal erythropoiesis.

Erythroblasts from Friend virus infected- and phenylhydrazine- treated mice accurately model erythroid differentiation

The dynamics of gene expression during terminal erythroid differentiation have been examined in three murine models; the erythroleukaemia cell line HCD‐57 and splenic erythroblasts isolated from mice treated with either the anaemia‐inducing strain of Friend virus (FVA cells) or the haemolytic agent phenylhydrazine (PHZ cells). In response to erythropoietin (EPO) and haemin, HCD‐57 cells proliferated and synthesized haemoglobin, but failed to complete terminal differentiation as indicated by lack of change in both gene expression and morphological appearance. In contrast, EPO‐induced terminal differentiation in FVA and PHZ cells in vitro was accompanied by increases in haemoglobin positivity, morphological maturation and a shared pattern of gene expression. EPO receptor (EPO‐R) mRNA levels peaked before globin gene expression which was maximal at 24 h. Peak GATA‐1 and EKLF mRNA levels also preceded the globin gene peak, but the highest NF‐E2 levels coincided with maximal globin levels, suggesting a role for NF‐E2 in the maintenance, rather than the initiation of globin gene expression. Peak expression of δ‐aminolaevulinic acid synthase (ALAS) coincided with peak globin expression. FVA and PHZ cells represent more effective models than the HCD‐57 cell line for the investigation of erythroid gene expression during EPO‐regulated terminal erythropoiesis.