Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7

Komatsu, Norio; Yamamoto, Masayuki; Fujita, Hiroko; Miwa, Akiyoshi; Hatake, Kiyohiko; Endo, Toshiyasu; Okano, Hiromitsu; Katsube, Takao; Fukumaki, Yasuyuki; Sassa, S.

doi:10.1182/blood.v82.2.456.bloodjournal822456

Cited by 70 publications

(49 citation statements)

References 33 publications

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“…To test whether EpoR mRNA was present, we isolated total RNA and subsequently performed RT‐PCR using primers to the human sequence of exon 3 and exon 7. As a positive control, we used the Epo‐dependent leukaemia cell line UT‐7, which is known to express the EpoR (Komatsu et al 1993). A fragment of the expected size of 485 bp was observed by agarose gel electrophoresis in both cell lines (Fig.…”

Section: Resultsmentioning

confidence: 99%

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

Assandri

Egger

Gassmann

et al. 1999

The Journal of Physiology

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Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK‐N‐MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. Expression of the EpoR in SK‐N‐MC cells was detected by reverse transcription‐PCR, Western blot and immunofluorescence analysis. Patch‐clamp studies of SK‐N‐MC cells confirmed the expression of T‐type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. Confocal laser scanning microscopy analysis of SK‐N‐MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T‐type Ca2+ channel blocker flunarizine. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T‐type voltage‐dependent Ca2+ channels.

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Section: Resultsmentioning

confidence: 99%

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

Assandri

Egger

Gassmann

et al. 1999

The Journal of Physiology

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“…The UT‐7/EPO cell line was maintained in liquid culture with Iscove's Modified Dulbecco's Medium (IMDM; GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and EPO (1 U/ml) (Komatsu et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

Activation of extracellular signal‐regulated kinases ERK1 and ERK2 induces Bcl‐xL up‐regulation via inhibition of caspase activities in erythropoietin signaling

Mori

Uchida

Watanabe

et al. 2003

Journal Cellular Physiology

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Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.

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“…It is worth noticing that growth of both original cell lines is not only supported by EPO but also shows dependency on GM‐CSF and IL‐3, both cytokines acting through their receptors constituted by a specific alpha subunit and the common beta subunit. However, when Komatsu et al 24 established the UT‐7/EPO subline, they reported that these cells had lost their response to GM‐CSF and IL‐3 due to a markedly decrease of the common beta subunit expression. Contrarily, TF‐1 cells exhibit complete growth dependency on those cytokines but EPO can only sustain short‐term growth.…”

Section: Resultsmentioning

confidence: 99%

“…The in vitro activity of EPO variants was determined by their ability to stimulate proliferation of UT‐7 and TF‐1 erythroid cell lines. 24, 25 Serial 1:2 dilutions of rhEPO or rhNEPO from 4 ng/mL to 0.01 ng/mL (UT‐7 cells) and from 15 ng/mL to 0.06 ng/mL (TF‐1 cells) were placed into culture microtiter plate wells. Cells were harvested from exponentially growing cultures, washed thrice with basal medium and resuspended in assay medium.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity

Mattio

Ceaglio

Oggero

et al. 2011

Biotechnology Progress

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Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side-effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms -designated as neuroepoetin (rhNEPO)- was purified to homogeneity from the supernatant of a CHO-producing cell line by a four-step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25-times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half-life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side-effects.

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Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7

Cited by 70 publications

References 33 publications

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

Activation of extracellular signal‐regulated kinases ERK1 and ERK2 induces Bcl‐xL up‐regulation via inhibition of caspase activities in erythropoietin signaling

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity

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