Erythropoietin-producing Cell Research Articles

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures invitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages invitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Time to Classify Tumours of the Stomach and the Kidneys According to Cell of Origin.

Malignant tumours are traditionally classified according to their organ of origin and whether they are of epithelial (carcinomas) or mesenchymal (sarcomas) origin. By histological appearance the site of origin may often be confirmed. Using same treatment for tumours from the same organ is rational only when there is no principal heterogeneity between the tumours of that organ. Organ tumour heterogeneity is typical for the lungs with small cell and non-small cell tumours, for the kidneys where clear cell renal carcinoma (CCRCC) is the dominating type among other subgroups, and in the stomach with adenocarcinomas of intestinal and diffuse types. In addition, a separate type of neuroendocrine tumours (NETs) is found in most organs. Every cell type able to divide may develop into a tumour, and the different subtypes most often reflect different cell origin. In this article the focus is on the cells of origin in tumours arising in the stomach and kidneys and the close relationship between normal neuroendocrine cells and NETs. Furthermore, that the erythropoietin producing cell may be the cell of origin of CCRCC (a cancer with many similarities to NETs), and that gastric carcinomas of diffuse type may originate from the ECL cell, whereas the endodermal stem cell most probably gives rise to cancers of intestinal type.

Abstract 2634: The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression

Abstract Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 varies among established prostate cancer cell lines. Nevertheless, we consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer. Citation Format: Marwah M. Al-Mathkour, Bekir Cinar. The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2634.

Erythropoietin-producing renal cell carcinoma arising from autosomal dominant polycystic kidney disease

Erythropoietin (EPO)-producing renal cell carcinomas (RCC) in patients with chronic renal failure secondary to autosomal dominant polycystic kidney disease (ADPKD) has not previously been reported. We report a case of EPO-producing RCC associated with ADPKD in a 66-year-old woman, and discuss the clinical and radiological findings.

Oxygen-regulated erythropoietin gene expression is dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Characterisation of functional domains within the mouse erythropoietin 3′ enhancer conveying oxygen-regulated responses in different cell lines

We have analysed sequences within the mouse erythropoietin enhancer which are required for oxygen regulated operation in the erythropoietin producing cell line, HepG2, and in two non-erythropoietin producing cell lines; the lung fibroblastoid cell line a23, and mouse erythroleukaemia (MEL) cells. At least three critical sites were demonstrated within a 96 nucleotide sequence. Oxygen regulated operation was dependent on sites within the first 26 nucleotides. Sequences lying 3′ to this region modulated enhancer function but did not themselves convey oxygen regulated operation. In HepG2 cells these 3′ sequences co-operated to permit operation of the inducible element at a distance from a promoter, but in MEL cells 3′ sequences repressed activity of the inducible element. Though operation of this 3′ sequence differed according to the cell type, oxygen regulated operation was dependent on the same two critical sites in the 5′ region in both erythropoietin producing and non-erythropoietin producing cells. These findings support the existence of a widespread oxygen sensing system in mammalian cells which is similar to that operating in specific cells to regulate erythropoietin production, and they indicate that the system activates factors with similar DNA sequence specificity in different cells.

Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures.

The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogenous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus nonhypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale.

Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taq-polymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121 degrees C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10(3) per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120 degrees C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.