Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2474-2474
Author(s):  
Jesus Duque-Afonso ◽  
Aitomi Essig ◽  
Leticia M Solari ◽  
Tobias Berg ◽  
Heike L. Pahl ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Functional Role ◽  
Target Gene ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

Flt3/ITD Blocks Myeloid Differentiation Of Hematopoietic Cells By Up-Regulating Runx1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3808-3808
Author(s):  
Tomohiro Hirade ◽  
Mariko Abe ◽  
Chie Onishi ◽  
Seiji Yamaguchi ◽  
Seiji Fukuda
Keyword(s):  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Cell Number ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Differentiation And Proliferation ◽  
Runx1 Expression ◽  
Novel Therapeutic

Abstract Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD. A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD+AML cells (N=78) than in FLT3/ITD-AML cells (N=190, P<0.05). The mRNA microarray analysis consistently demonstrated that Runx1 is up-regulated by Flt3/ITD in Ba/F3 cells. Up-regulation of Runx1 by Flt3/ITD was validated in Ba/F3 cells and 32D cells by quantitative RT-PCR. Incubation of control 32D cells with 20 ng/ml of G-CSF increased the number of Gr-1+/Mac-1+cells, whereas the induction of myeloid differentiation by G-CSF was abrogated by the overexpression of Flt3/ITD in 32D cells. By contrast, transduction of shRNA specific for Runx1 into Flt3/ITD+32D cells inhibited the expression of Runx1 mRNA by 60 % but increased the number and the proportion of Gr-1+/Mac-1+cells ; these effects were enhanced by incubation with G-CSF. These data indicate that Runx1 mediates the block of differentiation toward the myeloid lineage that is induced by Flt3/ITD. Moreover, the number of colony-forming units (CFU) over-expressing Flt3/ITD cultured in the absence of growth factors was reduced by Runx1-shRNA without affecting the total cell number in the suspension culture, as compared to Flt3/ITD+32D cells transduced with control-shRNA. This implies that antagonizing Runx1 facilitates the production of terminally differentiated cells that have lost colony-forming ability, thereby reducing the CFU number without altering the total number of cells. Finally, Runx1-shRNA inhibited the formation of secondary CFU colonies derived from the primary Flt3-ITD-over-expressing CFU colonies. Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD+cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD+cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD+cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD+hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD+AML cells in AML patients, in addition to inhibiting their aberrant proliferation. Disclosures: No relevant conflicts of interest to declare.

The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Kevin W. Harris ◽  
Xian-Jue Hu ◽  
Suzanne Schultz ◽  
Murat O. Arcasoy ◽  
Bernard G. Forget ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytoplasmic Domain ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Full Length ◽  
Cytokine Receptor ◽  
Myeloid Differentiation ◽  
Hematopoietic Growth Factors ◽  
Granulocytic Differentiation

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation. © 1998 by The American Society of Hematology.

scholarly journals Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2368-2368
Author(s):  
Luise A de Albuquerque Simoes ◽  
Isabel Weinhäuser ◽  
Diego A Pereira-Martins ◽  
César Alexander Ortiz Rojas ◽  
Thiago Mantello Bianco ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Functional Role ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Leukemic Cells ◽  
Control Group

Abstract Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML. Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p&lt;0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. Disclosures No relevant conflicts of interest to declare.

scholarly journals MiR-200c-3p aggravates gastric cell carcinoma via KLF6

2021 ◽  
Vol 43 (11) ◽  
pp. 1307-1316
Author(s):  
Ying Wang ◽  
Kaijuan Lu ◽  
Weibing Li ◽  
Zhigang Wang ◽  
Jing Ding ◽  
...  
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Functional Role ◽  
Migration And Invasion ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Gastric Cell

Abstract Background Gastric cell carcinoma (GCC) is a common and high-incidence malignant gastrointestinal cancer that seriously threatens human life and safety. Evidences suggest that microRNAs (miRNAs) exhibit an essential role in regulating the occurrence and development of GCC, while the effects and possible mechanisms remain to be further explored. Objective This study was designed to explore whether miR-200c-3p exerted its functional role in the growth and metastasis of GCC, and investigate the possible mechanisms. Methods The expression levels of miR-200c-3p in GCC tissues and cell lines were detected by qRT-PCR analysis. The functional role of miR-200c-3p in the viability, proliferation, migration and invasion of GCC cells were evaluated by CCK-8, EdU, wound healing and Transwell assays. In addition, the candidate targets of miR-200c-3p was predicted and confirmed by dual-luciferase reporter assay. Moreover, the relationship between miR-200c-3p and target (Krüppel like factor 6, KLF6) was assessed by qRT-PCR and western blot assays. Besides, the expression levels of KLF6 in GCC cells were determined by qRT-PCR and western blot assays. Furthermore, the role of KLF6 in the viability, proliferation, migration and invasion of GCC cells mediated with miR-200c-3p mimics was evaluated by CCK-8, EdU, wound healing and Transwell assays. Results In the present study, a new tumor promoting function of miR-200c-3p was disclosed in GCC. We found that the expression of miR-200c-3p was obviously increased in clinic GCC tissues and cell lines. In addition, down-regulation of miR-200c-3p suppressed cell viability, proliferation, migration, and invasion in GCC cells. Moreover, KLF6 was verified as a direct target of miR-200c-3p by binding its 3’-UTR. Additionally, KLF6 was remarkably decreased and was negatively associated with the miR-200c-3p expression in GCC cell lines. Furthermore, over-expression of KLF6 retarded the effects of miR-200c-3p on the growth and metastasis of GCC cell lines. Conclusions MiR-200c-3p potentially played a tumor-promoting role in the occurrence and development of GCC, which may be achieved by targeting KLF6. Graphic abstract

The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Kevin W. Harris ◽  
Xian-Jue Hu ◽  
Suzanne Schultz ◽  
Murat O. Arcasoy ◽  
Bernard G. Forget ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytoplasmic Domain ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Full Length ◽  
Cytokine Receptor ◽  
Myeloid Differentiation ◽  
Hematopoietic Growth Factors ◽  
Granulocytic Differentiation

Abstract The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation. © 1998 by The American Society of Hematology.

Enforced Expression of Mir-125b Blocks Granulocytic Differentiation by Simultaneous Repression of Multiple Targets.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1554-1554
Author(s):  
Ewa Surdziel ◽  
Maciej Cabanski ◽  
Iris Dallmann ◽  
Arnold Ganser ◽  
Michaela Scherr ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation ◽  
Multiple Targets ◽  
Knock Down

Abstract Abstract 1554 Granulopoiesis is a multistep process controlled by a complex system of cytokines and transcription factors that modulate expression of downstream genes and mediate proliferation and differentiation signals. Recent findings demonstrate that miRNAs may provide an additional level of control. We used the 32D murine myeloid progenitor cell line as a model system to study G-CSF-induced granulocytic differentiation. Based on miRNA-expression analyzed by microarray and miR-qRT-PCR, we identified several miRNAs (including miR-34a-c, -125b, -155, 181b, 223, 291a, 370) potentially involved in regulation of granulocytic differentiation. To define the role of individual miRNAs, stable gain- and loss-of-function phenotypes were generated using lentiviral gene transfer of pre-miRNAs and antagomiRs, respectively. We found that enforced expression of miR-125b in undifferentiated 32D myeloid precursors resulted in a complete block of granulocytic differentiation upon G-CSF treatment and partially protected the cells from IL-3 withdrawal-induced cell death. The pivotal role of miR-125b in myeloid differentiation was demonstrated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with the chromosomal translocation t(2;11)(p21;q23) resulting in miR-125b over-expression [Bousquet M, et al. JEM 2008]. Furthermore, clonogenic assays of primary Lin- cells revealed that miR-125b cultures generated more and larger colonies as compared to miR-control, indicating a proliferative advantage of miR-125b over-expressing progenitor cells. Correspondingly, enforced miR-125b expression in murine bone marrow has recently been shown to induce a lethal myeloproliferative disorder (MPD) in transplanted mice [O'Connell RM, et al. PNAS 2010]. However, the molecular mechanisms mediated by miR-125b in hematopoietic cells remain largely unknown. By utilizing different miRNA-target prediction programs including Target Scan, DIANA microT v 0.3 and RNA22, we identified Bcl-2 family members Bak1, Mcl-1, Bmf and Puma as putative targets of miR-125b. We confirmed diminished Bak1 protein expression in 32D cells (reduction by ∼40%); however, lentivirus-mediated RNAi targeting Bak1 to the similar level as induced by enforced miR-125b expression resulted only in a delay of cell death in the presence of G-CSF but not in granulocytic differentiation block. Further computational analysis revealed two putative miR-125b binding sites in 3’ UTR of Stat3, the principal Stat protein activated following G-CSF treatment and robustly involved in myeloid differentiation. Specific binding of miR-125b to Stat3 3’UTR was validated by luciferase reporter assay and confirmed by western blotting (reduction by ∼30-40%). We found that shRNA-mediated strong reduction of Stat3 protein (reduction by ∼80%) enables G-CSF dependent cell proliferation (with a delay of some days), and induces a complete block of cellular differentiation. On the contrary, a mild reduction of Stat3 protein expression(by ∼30–40%), as induced by enforced expression of miR-125b, resulted only in a slight delay of cell death but not in block of granulocytic differentiation. Interestingly, simultaneous reduction of both Bak1 and Stat3 by combinatorial RNAi to similar levels as observed in the presence of enforced miR-125b expression, cooperatively delays cell death upon G-CSF treatment as compared to knock-down of Bak1 or Stat3 solely. Since miR-125b has approximately 500 predicted target genes and neither mild knock-down of Bak1 nor Stat3 alone resulted in a block of granulocytic differentiation, we may either miss an additional/unknown important target or the miR-125b-induced phenotype requires simultaneous repression of more targets in addition to Stat3 and Bak1. Nevertheless our study provides experimental evidence that miR-125-mediated phenotypes arise from mild and simultaneous down-regulation of multiple targets/signalling pathways. Therefore the precise and quantitative analysis of miRNA-targets is required to evaluate the safety and benefit of eventual miRNA-based therapeutic strategies to modulate complex cellular phenotypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nonhistone protein antigen profiles of five leukemic cell lines reflect the extent of myeloid differentiation

Blood ◽  
1984 ◽  
Vol 63 (3) ◽  
pp. 701-710 ◽  
Author(s):  
A Goldberger ◽  
G Brewer ◽  
LS Hnilica ◽  
RC Briggs
Keyword(s):  
Cell Lines ◽  
Myeloid Cell ◽  
Leukemic Cell ◽  
Nuclear Extract ◽  
Myeloid Differentiation ◽  
Chromosomal Protein ◽  
Protein Antigens ◽  
Nonhistone Protein ◽  
Leukemic Cell Lines ◽  
Chromatin Proteins

Abstract The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS- polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

scholarly journals Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines

Leukemia ◽  
2002 ◽  
Vol 16 (4) ◽  
pp. 683-692 ◽  
Author(s):  
MB Miranda ◽  
TF McGuire ◽  
DE Johnson
Keyword(s):  
Cell Lines ◽  
Myeloid Cell ◽  
Granulocytic Differentiation

scholarly journals Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3902-3908 ◽  
Author(s):  
Q Shen ◽  
S Chada ◽  
C Whitney ◽  
PE Newburger
Keyword(s):  
Steady State ◽  
Glutathione Peroxidase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Dimethyl Formamide ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Regulation Of Expression

We have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL- 60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.

scholarly journals MicroRNA-23a promotes colorectal cancer cell migration and proliferation by targeting at MARK1

2019 ◽  
Vol 51 (7) ◽  
pp. 661-668 ◽  
Author(s):  
Xiaoli Tang ◽  
Meiyuan Yang ◽  
Zheng Wang ◽  
Xiaoqing Wu ◽  
Daorong Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Functional Role ◽  
Expression Level ◽  
Colorectal Cancer Cell ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Proliferation And Migration

Abstract The functional role of microRNA-23a in tumorigenesis has been investigated; however, the exact mechanism of microRNA-23a (miR-23a) in colorectal cancer development has not been fully explored. In the present study, we aimed to investigate the molecular functional role of miR-23a in colorectal carcinogenesis. Quantitative real-time polymerase chain reaction was conducted to investigate the expression level of miR-23a in tissue samples and cell lines (HCT116 and SW480). CCK-8, colony formation and Transwell assay were used to explore the role of miR-23a in cell proliferation and migration. Dual luciferase reporter assay was used to identify the direct binding of miR-23a with its target, MARK1. Western blot analysis was used to analyze the expression level of MARK1, as well as a confirmed miR-23a target gene, MTSS1, in miR-23a-mimic and miR-23a-inhibit groups. Rescue experiments were conducted by overexpression of MARK1 in miR-23a-mimic-transfected cell lines. The results showed that miR-23a was highly expressed in colorectal cancer tissue and cell lines. MiR-23a could promote proliferation and migration of colorectal cancer cell lines. MARK1 was a direct target of miR-23a and the expression level of MARK1 was down-regulated in miR-23a-mimic-transfected cell lines but up-regulated in miR-23a-inhibit-transfected cells. Overexpression of MARK1 could partly reverse the cancer-promoting function of miR-23a. Our results suggested that miR-23a promotes colorectal cancer cell proliferation and migration by mediating the expression of MARK1. MiR-23a may be a potential therapeutic target for colorectal cancer treatment.

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