Erythropoietin, erythropoiesis and beyond.

Erythropoietin (EPO) is a glycoprotein that is mainly produced in the adult kidney, and it was initially highlighted for its action on the hematopoietic system. Moreover, EPO is also expressed in several non-hematopoietic tissues, where it plays a role in the protection from apoptosis and inflammation due to hypoxia, toxicity or injury. These protective effects are mainly known and studied in cardioprotection and neuroprotection but are also reported in retina degeneration, auditory injury and pancreatic-related diseases. The tissue protective effect of EPO is mainly mediated through the interaction with the heterodimeric receptor EPOR/ßcR. Human recombinant EPO (HuREPO), which has been developed to treat anemia, is not adequate for tissue protection. The low affinity of the alternative receptor for EPO involves the injection of excessive concentration of erythropoiesis-stimulating agents (ESAs), implicating side effects due to the cross-talk with hematopoietic activity. For these reasons, EPO derivatives with less affinity for the EPO homodimeric receptor are under development. In this review, we provide an overview of the erythroid and non-erythroid functions of EPO by detailing the molecular mechanisms activated by the binding of EPO to its receptors in different tissues.

Pro-inflammatory cytokine-mediated anemia: regarding molecular mechanisms of erythropoiesis.

Anemia of cancer and chronic inflammatory diseases is a frequent complication affecting quality of life. For cancer patients it represents a particularly bad prognostic. Low level of erythropoietin is considered as one of the causes of anemia in these pathologies. The deficiency in erythropoietin production results from pro-inflammatory cytokines effect. However, few data is available concerning molecular mechanisms involved in cytokine-mediated anemia. Some recent publications have demonstrated the direct effect of pro-inflammatory cytokines on cell differentiation towards erythroid pathway, without erythropoietin defect. This suggested that pro-inflammatory cytokine-mediated signaling pathways affect erythropoietin activity. They could interfere with erythropoietin-mediated signaling pathways, inducing early apoptosis and perturbing the expression and regulation of specific transcription factors involved in the control of erythroid differentiation. In this review we summarize the effect of tumor necrosis factor (TNF)alpha, TNF-related apoptosis-inducing ligand (TRAIL), and interferon (IFN)-gamma on erythropoiesis with a particular interest for molecular feature.

Curcumin-induced cell death in two leukemia cell lines: K562 and Jurkat.

Curcumin presents strong antioxidant and anticancer properties. However, molecular mechanisms leading to curcumin-induced cell death are poorly understood. The effect of curcumin was compared in two different leukemia cell lines: K562 and Jurkat. Cell death was induced in both cell lines, and apoptosis pathways were investigated by Western blot analysis. Decreases in pro-caspase 8 and 9 levels were observed. BH(3) interacting domain death agonist (Bid) was also cleaved. Jurkat cells appeared to be more sensitive to curcumin, and apoptosis takes place earlier.

Regulation of transcription of the glutathione S-transferase P1 gene by methylation of the minimal promoter in human leukemia cells.

To study the relationship between methylation and the transcriptional activity of the minimal promoter of the glutathione S-transferase GSTP1 gene encoding glutathione S-transferase P1-1, GSTP1 mRNA levels as well as basal promoter activity were compared in human leukemia cell lines. The K562 erythroleukemia cell line presented a strong GSTP1 promoter activity, as measured in transient transfection assays using a luciferase reporter plasmid, and correlated with a high mRNA whereas in Raji cells no mRNA was expressed. In order to establish a relationship between the expression and the methylation status, we used in vitro bisulfite sequencing which indicated that both methylated and unmethylated GSTP1 promoter alleles coexisted in K562 cells, whereas Raji lymphoma cells showed a nearly uniform hypermethylation of the promoter region. To determine the impact of methylation, we used in vitro SssI methylation of the minimal GSTP1 promoter, which led to the silencing of the promoter activity in transient transfection assays in expressing K562 as well as in non-expressing Raji cells. These data are in good agreement with previously obtained results and indicate that methylation of CpG sites of the basal promoter is an essential mechanism in the control of GSTP1 gene expression in human leukemia.

Phorbol ester responsiveness of the glutathione S-transferase P1 gene promoter involves an inducible c-jun binding in human K562 leukemia cells.

Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells. However, the mechanisms of the transcriptional activation of this gene remain unclear. In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12-O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription. Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA. By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex. These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model.

GTP-mediated differentiation of the human K562 cell line: transient overexpression of GATA-1 and stabilization of the gamma-globin mRNA.

Induction of specific gene expression may provide an alternative or a support to conventional cytotoxic chemotherapy of cancer, as well as to therapy for sickle cell diseases. In this respect, pharmacological induction of expression of the endogenous gamma-globin gene is a realistic approach to therapy of beta-globin disorders. Erythroid differentiation and inhibition of proliferation of the human CML K562 cell line was induced by guanosine 5'-triphosphate (GTP). The hemoglobin production in cells was correlated to an increase in alpha- and gamma-globin mRNA expression. At the transcriptional level, we showed that both the expression of the major erythroid transcription factor GATA-1 (protein and mRNA) and its binding capacity to the gamma-globin gene promoter was transiently increased. Moreover, GTP moderately stimulated the gamma-globin gene promoter after 48 h of treatment. At the post-transcriptional level, GTP treatment led to a drastic increase of the gamma-globin mRNA half-life. This stabilizing effect of GTP was mediated via the 3'-untranslated region (3'-UTR) of the gamma-globin mRNA. In conclusion, mechanism of GTP-mediated differentiation of K562 cells is linked to an early activation of gamma-globin gene transcription followed by a stabilization of its mRNA.

Anthracyclines as tumor cell differentiating agents: effects on the regulation of erythroid gene expression.

Tumor cells, and particularly leukemic cells, can be considered as maturation-arrested cells which have escaped some normal control and continue to proliferate. This maturation arrest can be reversed by differentiation agents such as antitumor drugs currently used in conventional cytotoxic chemotherapy. In this respect, anthracyclines have been shown to trigger the differentiation of leukemic and solid tumor cells, but the molecular mechanisms by which such drugs lead to the differentiating phenotype are still poorly understood. Using human leukemic multipotent K562 cells, we have demonstrated that subtoxic concentrations of aclacinomycin (ACLA) and doxorubicin (DOX) preferentially stimulate the hemoglobinic pathway (globins and heme synthesis) and the expression of mRNAs of globins and of porphobilinogen deaminase (PBGD). However, our results indicate that both drugs exert this differentiating effect along distinct regulatory pathways. Indeed, only ACLA and not DOX induces the expression of erythropoietin receptor (EpoR) mRNAs and of membrane EpoR, as well as an overexpression of the erythroid transcription factors GATA-1 and NF-E2 known to play a central role in erythroid gene regulation. Similarly, using transfection assays, ACLA but not DOX activates the regulatory regions (promoters and enhancers) of GATA-1, EpoR, PBGD, epsilon- and gamma-globin genes. Finally, results of run-on assays indicate that ACLA induces an enhancement of the transcription rate of these erythroid genes whereas DOX preferentially increases stability of GATA-1, NF-E2 and PBGD mRNAs. In conclusion, ACLA mainly acts at the transcriptional level via specific activation of erythroid regulatory regions whereas DOX rather acts at the posttranscriptional level by increasing the half-lives of erythroid mRNAs.

Transcriptional and posttranscriptional regulation of erythroid gene expression in anthracycline-induced differentiation of human erythroleukemic cells.

Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX.

Evidence for distinct regulation processes in the aclacinomycin- and doxorubicin-mediated differentiation of human erythroleukemic cells.

Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.

Increased expression of GATA-1 and NFE-2 erythroid-specific transcription factors during aclacinomycin-mediated differentiation of human erythroleukemic cells.

Anthracycline antitumor drugs, particularly aclacinomycin (ACM) have been shown to be potent inducers of erythroid differentiation in human leukemic K562 cells. Here we report that such an event is associated with an overexpression of the erythroid-specific transcription factors GATA-1 and NFE-2. Using the electrophoretic mobility shift assay, during differentiation over 3 days of culture, we have observed an increase in the binding either of GATA-1 to the promoter of the gamma-globin gene (region -201 to -156) or NFE-2 to the promotor of the porphobilinogen deaminase gene (region -170 to -142). Both events were paralleled by a recruitment of hemoglobinized cells and a stimulation of heme synthesis. Enhanced binding capacity of GATA-1 was confirmed by an increase in its mRNAs. Moreover, GATA-1 and NFE-2 overexpression has been shown to be specific of the differentiating effect of the drug and not of its growth inhibitory effect. In contrast, no change was observed in the binding of the ubiquitous factors OTF-1 and AP-1, except on day 3, where AP-1 decreased. Although ACM is a DNA-intercalating agent, it did not directly affect transcription factors binding to their cis-sequences as assessed by the preincubation of the oligonucleotides probes with increasing concentrations of ACM. Taken together, these results strongly suggest that ACM could exert their erythroid-differentiating activity by modulating the expression of transcription factors which specifically regulate the transcription of erythroid genes.