scholarly journals Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 263-268 ◽  
Author(s):  
WD Hankins ◽  
J Schooley ◽  
C Eastment
Keyword(s):  
Cell Lines ◽  
Single Cells ◽  
Gene Activation ◽  
Active Agent ◽  
Erythroid Cell ◽  
Biological Assays ◽  
Erythroid Precursors ◽  
Erythroleukemia Cell

Abstract Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.

scholarly journals Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 263-268
Author(s):  
WD Hankins ◽  
J Schooley ◽  
C Eastment
Keyword(s):  
Cell Lines ◽  
Single Cells ◽  
Gene Activation ◽  
Active Agent ◽  
Erythroid Cell ◽  
Biological Assays ◽  
Erythroid Precursors ◽  
Erythroleukemia Cell

Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

Abstract To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

scholarly journals KLF1 Acts As a Pioneer Transcription Factor to Open Chromatin and Facilitate Recruitment of GATA1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 501-501
Author(s):  
Kevin R. Gillinder ◽  
Graham Magor ◽  
Charles Bell ◽  
Melissa D. Ilsley ◽  
Stephen Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Cell Lines ◽  
Zinc Finger Protein ◽  
Gene Activation ◽  
The Body ◽  
Erythroid Cell ◽  
Small Subset ◽  
Open Chromatin

Abstract Only a small subset of transcription factors (TFs) can act as pioneer factors; i.e. those that can 'open' otherwise 'closed' chromatin to facilitate assembly of TF complexes and co-factors to enable transcription. The KLF/SP family of TFs bind to a 9-10 bp consensus motif in DNA to activate or repress target gene expression. We have studied the potential for KLF1, which is essential for erythropoiesis, to provide a pioneering function in erythroid progentior cells. Previous ChIP-seq studies have shown KLF1 binds a few thousand enhancers and promoters to activate erythroid cell gene expression 1. It often binds near to other key erythroid TFs such as GATA1 and SCL/TAL1, so is likely to work in concert with them in some contexts. We have employed an inducible stable KLF1-ERTM construct to rescue gene expression and differentiation of Klf1-/- erythroid cell lines 2. We employed ChIP-seq, ATAC-seq and DNAse1 HS to show KLF1 can bind to closed sites in chromatin and induce an open state. We show this is essential for recruitment of the settler transcription, GATA1, at certain co-bound sites but not others. This pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such the one at -26kb in the a-globin LCR and one within the body of the E2f2 gene 3 but rarely at promoters. We further show that two different neomorphic mutations in the KLF1 DNA-binding domain lead to ectopic pioneering (opening of closed chromatin) and aberrant gene activation 4. We generated a series of N-terminal deletions in KLF1 and employed ATAC-seq to map the domain/s within KLF1 responsible for the pioneering activity and show it is distinct from DNA-binding activity. The domain is responsible for bromodomain protein recruitment, the likely effector of chromatin remodelling. We have also examined whether KLF3, which acts as a transcription repressor via recruitment of the co-repressor, CtBP2, can force the closure of otherwise open chromatin 5. We find it cannot. Rather, KLF3 (and likely other members of this subclade) works via active recruitment of co-repressors rather than rendering chromatin inaccessible. This likely enables rapid reactivation of pioneered enhancers without the need to reprogram chromatin. This work has broad implications for how the KLF/SP family of TFs work in vivo to reprogram cells and direct differentiation. We will present data for such activity in non-erythroid cell systems. References:Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063.Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868.Tallack MR, Keys JR, Humbert PO, Perkins AC. EKLF/KLF1 controls cell cycle entry via direct regulation of E2f2. J Biol Chem. 2009;284(31):20966-20974.Gillinder KR, Ilsley MD, Nebor D, et al. Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability. Nucleic Acids Res. 2017;45(3):1130-1143.Turner J, Crossley M. Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators. Embo J. 1998;17(17):5129-5140. Disclosures Perkins: Novartis Oncology: Honoraria.

scholarly journals LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i7-i7
Author(s):  
Jiaojiao Deng ◽  
Sophia Chernikova ◽  
Wolf-Nicolas Fischer ◽  
Kerry Koller ◽  
Bernd Jandeleit ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Tumors ◽  
Cell Lines ◽  
Chemotherapeutic Agent ◽  
Leptomeningeal Metastasis ◽  
Breast Cancer Cell Lines ◽  
Normal Brain ◽  
Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Farnaz Dabbagh Moghaddam ◽  
Iman Akbarzadeh ◽  
Ehsan Marzbankia ◽  
Mahsa Farid ◽  
Leila khaledi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Treatment ◽  
Cell Lines ◽  
Encapsulation Efficiency ◽  
Inbred Mice ◽  
Breast Cancer Treatment ◽  
Anticancer Effect ◽  
Anti Cancer

Abstract Background Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, melittin, melittin-loaded niosome, and empty niosome had been optimized and the anticancer effect assessed in vitro on 4T1 and SKBR3 breast cell lines and in vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the niosomes; different niosomal formulations of melittin were prepared and characterized in terms of morphology, size, polydispersity index, encapsulation efficiency, release kinetics, and stability. A niosome was formulated and loaded with melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of melittin, and melittin-loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, flow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the gene expression. 40 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results This study showed melittin-loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation efficiency, PDI, and release rate and shows a high anticancer effect on cell lines. The melittin-loaded niosome affects the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. They have also enhanced the apoptosis rate and inhibited cell migration, invasion in both cell lines compared to the melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in melittin-loaded niosome. Renal and hepatic biomarker activity did not significantly differ in melittin-loaded niosome and melittin compared to healthy control. In immunohistochemistry, P53 expression did not show a significant change in all groups. Conclusions Our study successfully declares that melittin-loaded niosome had more anti-cancer effects than free melittin. This project has demonstrated that niosomes are suitable vesicle carriers for melittin, compare to the free form.

scholarly journals Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Jo Rademacher ◽  
Anahi Cruz ◽  
Mary Faber ◽  
Robyn A. A. Oldham ◽  
Dandan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Lines ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Murine Models ◽  
Lentiviral Transduction ◽  
Ifn Γ ◽  
Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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