Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2161-2161
Author(s):  
Jaira F. de Vasconcellos ◽  
Y. Terry Lee ◽  
Colleen Byrnes ◽  
Laxminath Tumburu ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Hemoglobin ◽  
Cell Counting ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Empty Vector ◽  
Gene And Protein Expression

Abstract HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Targeted Reduction of Let-7a miRNA Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Jaira F. de Vasconcellos ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
Joshua M. Allwardt ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mirna Family ◽  
Fetal Hemoglobin ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Total Hemoglobin ◽  
Cd34 Cells ◽  
Mrna Expression Levels

Abstract MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

LIN28A-Mediated Expression Of Fetal Hemoglobin Ameliorates Erythrocyte Sickling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 313-313
Author(s):  
Jaira F. de Vasconcellos ◽  
Ross M. Fasano ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
Colleen Byrnes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Transgenic Expression ◽  
Sickle Hemoglobin ◽  
Gamma Globin ◽  
Alpha Globin ◽  
And Control

Abstract Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes. After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009). These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures: No relevant conflicts of interest to declare.

The prognostic value of cytokeratin and extracellular collagen expression in urinary bladder cancer

2021 ◽  
Vol 21 ◽  
Author(s):  
Marc Ingenwerth ◽  
Péter Nyirády ◽  
Boris Hadaschik ◽  
Tibor Szarvas ◽  
Henning Reis
Keyword(s):  
Bladder Cancer ◽  
Protein Expression ◽  
Urinary Bladder Cancer ◽  
High Expression ◽  
Low Grade ◽  
Mrna Levels ◽  
High Grade ◽  
Expression Levels ◽  
Gene And Protein Expression ◽  
Muscle Invasive

Background: Expression levels of collagens have been implicated in the progression of various cancers and interact with cytokeratins, but are not well studied in bladder cancer (BC). Therefore, we analyzed the gene and protein expression levels of collagen 1A1 (Col1a1/COL1A1), collagen 3A1 (col3a1/COL3A1), collagen 5A2 (col5a2/COL5A2), cytokeratin 14 (krt14/CK14), and cytokeratin 17 (krt17/CK17) in urothelial BC samples of different stages. Methods: In total, 102 fresh frozen and 190 formalin fixed and paraffin embedded (FFPE) samples were tested using immunohistochemistry and RT-qPCR. Expression levels were correlated to clinicopathological and follow-up data. Results: Col1a1, col3a1, col5a2 and krt14 mRNA levels were significantly overexpressed in high-grade and muscle-invasive BC (MIBC) compared to low-grade and non-muscle invasive BC (NMIBC) cases. Disease-specific survival (DSS) was shorter in patients with high expression levels of col1a1 (p = 0.004), col3a1 (p = 0.004), and col5a2 (p = 0.028). CK14 (p = 0.020), COL3A1 (p = 0.006) and Col5A2 (p = 0.006) protein expression levels were significantly higher and protein expression levels of CK17 (p = 0.05) significantly lower in MIBC compared to NMIBC. Furthermore, CK14 (p = 0.002) and COL5A2 (p = 0.006) protein expressions were significantly higher in high-grade compared to low-grade BC. DSS was shorter in patients with high expression levels of COL5A2 (p = 0.033) and CK14 (p = 0.042). Conclusion: Expression changes of collagens and cytokeratins are univariable prognostic markers in BC.

scholarly journals Effect of EPA on Neonatal Pig Sertoli Cells “In Vitro”: A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies

2021 ◽  
Vol 12 ◽  
Author(s):  
Iva Arato ◽  
Veronica Ceccarelli ◽  
Francesca Mancuso ◽  
Catia Bellucci ◽  
Cinzia Lilli ◽  
...  
Keyword(s):  
Protein Expression ◽  
Sertoli Cells ◽  
Fold Change ◽  
Control Group ◽  
Mrna Levels ◽  
Expression Levels ◽  
Chemotherapy Drugs ◽  
Gene And Protein Expression ◽  
Pubertal Boys

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 μM vs SCs and ***p < 0.001 at 3.33μM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 μM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 μM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 50-50
Author(s):  
Y. Terry Lee ◽  
Colleen Byrnes ◽  
Jaira F. de Vasconcellos ◽  
Megha Kaushal ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Counting ◽  
Specific Gene ◽  
Globin Mrna ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Gamma Globin ◽  
Gene And Protein Expression

Abstract LIN28 proteins bind to RNA and regulate developmental timing events in multicellular organisms, in part, by reducing cellular levels of the let-7 family of microRNAs. High-level LIN28 expression in stem cells promotes their self-renewal. Over-expression of the LIN28 proteins causes suppression of let-7 in hematopoietic stem and early progenitor cell populations (CD34+) from adult donors and manifests a more fetal-like phenotype in the erythroid lineage. Here we explore LIN28expression that is restricted to erythroid cells, rather than stem or multi-potential progenitor cells. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes, as well as an internal ribosomal entry site for puromycin selection (vectors: KLF1-LIN28A-OE and SPTA1-LIN28A-OE). Viral supernatants from these constructs were compared with empty-vector controls in matched transductions of CD34+ cells from three adult human volunteers. The cells were transduced and cultured using a three-phase, serum-free model for ex vivo erythropoiesis. Erythroblast proliferation and differentiation were comparable between control and LIN28-transduced cells assessed by cell counting and flow cytometry with staining for CD71, glycophorin A and thiazole orange. To validate restricted expression of LIN28 in the erythroid lineage, colony formation assays were performed in semisolid methylcellulose containing 1.0 ug/ml puromycin. BFU-E, CFU-GM, CFU-G, CFU-M and GEMM colonies were enumerated 14 days after plating. Puromycin addition to KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions resulted in selection of the erythroid colonies (BFU-E as a percentage of total colonies: Control: 44.6 ± 6.1%; KLF1-LIN28A-OE: 98.4 ± 0.7%, p=0.003; SPTA1-LIN28A-OE: 95.2 ± 1.1%, p=0.005). LIN28A over-expression was confirmed by RT-QPCR (KLF1-LIN28A-OE: 2.1E+05 ± 7.0E+04 copies/ng; SPTA1-LIN28A-OE: 2.2E+05 ± 8.3E+04 copies/ng; Controls: below detection limits) and Western analyses after transduction. Suppression of all let-7 miRNA family members to less than 30% control levels were detected for both vectors resulting in a reduction in total let-7 miRNA (RT-QPCR: Control: 2.0E+07 ± 9.7E+05 copies/ng; KLF1-LIN28A-OE: 5.6E+06 ± 5.6E+05 copies/ng, p=0.003; SPTA1-LIN28A-OE: 4.6E+06 ± 6.2E+05 copies/ng, p=0.003). BCL11A expression levels were also measured by RT-QPCR and Western analyses. While BCL11A showed no significant change at the mRNA level (Control: 1.2E+03 ± 4.5E+02 copies/ng; KLF1-LIN28A-OE: 2.9E+02 ± 7.4E+01 copies/ng, p=0.07; SPTA1-LIN28A-OE: 4.2E+02 ± 3.3E+02 copies/ng, p=0.07), protein analyses of nuclear BCL11A showed moderately reduced levels after KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions. Globin mRNA and protein levels were investigated and compared with controls. Gamma-globin mRNA was significantly increased in LIN28A-OE samples (Control: 3.6E+06 ± 8.2E+05 copies/ng; KLF1-LIN28A-OE: 1.9E+07 ± 1.7E+06 copies/ng, p=0.007; SPTA1-LIN28A-OE: 1.7E+07 ± 8.9E+05 copies/ng, p=0.003). Fetal hemoglobin (HbF) production was measured at the end of the culture period using High Performance Liquid Chromatography, and was increased in the KLF1-LIN28A-OE and SPTA1-LIN28A-OE samples compared to the control (Control: 7.0 ± 1.4%; KLF1-LIN28A-OE: 31.9 ± 2.7%, p=0.004; SPTA1-LIN28A-OE: 43.0 ± 6.2%, p=0.004). Flow cytometry analyses demonstrated a pan-cellular HbF distribution. In contrast to promoting self-renewal in stem cells, these data suggest that adult erythroblast-restricted LIN28 functions to partially reverse the fetal-to-adult developmental transition in hemoglobin expression. Disclosures No relevant conflicts of interest to declare.

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 387-393 ◽  
Author(s):  
Natarajan V. Bhanu ◽  
Tiffany A. Trice ◽  
Y. Terry Lee ◽  
Nicole M. Gantt ◽  
Patricia Oneal ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Synergistic Effects ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Mrna ◽  
Erythroid Precursor ◽  
Gamma Globin ◽  
Adult Human ◽  
Gene And Protein Expression ◽  
Complete Reversal

Abstract We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

Elevation of Fetal Hemoglobin (HbF) by Novel Alkylating Agents in Human Erythroid Cells; Synergistic Effects with Established HbF-Inducing Agents.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3815-3815
Author(s):  
I. Chiotoglou ◽  
M. Samara ◽  
S. Likousi ◽  
S. Samara ◽  
Z. Iakovidou ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Alkylating Agents ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Cell Number ◽  
Mrna Levels ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Mrna Transcripts

Abstract Pharmacological induction of fetal hemoglobin (HbF) is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia, mainly by hydroxyurea (HU). However, refractoriness or poor response of some patients treated with HU triggered research for other drugs. In the present study, we evaluated the effects of novel steroidal alkylating agents EA80, XK4 and CS on HbF induction in CD34+ cell cultures from normal donors. Furthermore, we examined the effects of these agents combined with HU, hemin (HE) and butyric acid (BU) on HbF modulation in adult erythroid cells. CD34+ cells from normal donors cultured in serum-free StemSpan medium were exposed to EPO (4 u/ml) + SCF (100ng/ml). Different concentrations of EA80 (0.1–1μM), XK4 (0.1–10 μM) and CS (0.1–1μM) were added to the cultures at day 6 (proerythroblast stage), then cells were washed and harvested 1– 3 days later. The effect of the drugs on cell number was measured by the trypan blue exclusion technique. The number of Hb-containing cells were determined using the benzidine-HCl procedure. EA80, over a wide range of concentrations (0.1–0.8μM), did not compromise cell survival. Continuous exposure of CD34+ to EA80 had a dose (up to 0.4μM)- and-time dependent effect on cell number as well as on globin mRNA levels. Treatment of CD34+ cells with EA80 at 0.4μM for 3 days was followed by a two-fold increase in cell number and a 1.5-fold in benzidine-positive cells. Qualitative and quantitative RT-PCR evaluation of globin-mRNA transcripts in CD34+ demonstrated that EA80 (0.4μM) caused a time- and dose-dependent increase in gamma globin mRNA (1– 3 days: 1.5 to 2.0-fold). The addition of HU and HE in combination with EA80 in normal CD34+ cell cultures led to a 20–30% increase in cell number by day 9. Furthermore, the combination of EA80 with either HU or HE was accompanied by an increase in γ-mRNA content (1.5 and 2.5-fold, respectively).No significant difference was detected in the level of both adult globin mRNAs. In contrast, BU addition had no effect either on erythroid cell proliferation or γ-mRNA levels. The addition of XK4 had a dose dependent effect on cell number and γ-globin mRNA transcripts [highest effect (2.0–5.0-fold) at 5 and 10μM]. Addition of EA80 (0.4μM) concurrently with varied concentrations of XK4 (0.1–10μM) caused a x2–4-fold increase in gamma globin content (highest effect at 5/10μM). For both drugs, the levels of β-and α-mRNAs in normal CD34+ cell cultures were not affected by dose. The addition of the third drug, CS at concentrations between 0.1 and 1μM proved toxic (reduced cell number and γ-globin mRNA transcripts). Our findings suggest that the beneficial effect of EA80 and XK4 might be threefold: increasing cell number affecting preferentially the rate of transcription of γ-globin mRNA, acting synergisticallly with HE and HU, most likely through transcriptional and posttranscriptional mechanisms. These results indicate that novel alkylating agents EA80 and XK4, either alone or in combination with other HbF-augmenting drugs, might provide a potentially useful treatment for patients with β-hemoglobinopathies with poor or no response to established Hb-F inducing agents.

Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α

2018 ◽  
Vol 18 (7) ◽  
pp. 1025-1031
Author(s):  
Cheng Luo ◽  
Di Wu ◽  
Meiling Chen ◽  
Wenhua Miao ◽  
Changfeng Xue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Confocal Microscopy ◽  
Protein Expression ◽  
Mtt Assay ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Gene And Protein Expression ◽  
Simulated Hypoxia

Background: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. Methods: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results： ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours’ relatively hypoxia incubation. Conclusion: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly

scholarly journals Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qilu Wei ◽  
Ning Kong ◽  
Xiaohui Liu ◽  
Run Tian ◽  
Ming Jiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fast Green ◽  
Expression Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

scholarly journals Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1862-1869 ◽  
Author(s):  
P Constantoulakis ◽  
B Nakamoto ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Cell Cultures ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Fetal Sheep ◽  
Gamma Globin

Abstract Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

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