C-myb mRNA Expression Research Articles

MiR-150 regulates B lymphocyte in autoimmune hemolytic anemia/Evans syndrome by c-Myb.

The objective of the study was to study the regulation of B lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome. From October 2015 to May 2016, 35 patients with AIHA/Evans in the Department of Hematology, Tianjin Medical University General Hospital were enrolled into this study. c-Myb mRNA and miR-150 expression in B lymphocytes were measured using real-time PCR (RT-PCR). Correlation between c-Myb and miR-150 and clinical parameters in patients with AIHA/Evans were analyzed. c-Myb mRNA expression in hemolysis patients was significantly higher than in remission patients and CLL patients, negatively correlated with hemoglobin (Hb) level and complement 3(C3) levels, and positively correlated with total bilirubin (TBIL) concentration and indirect bilirubin (IBIL) concentration. miR-150 expression in hemolysis patients was significantly lower than in patients in remission and CLL patients. miR-150 was negatively correlated with TBIL and IBIL, and positively correlated with Hb, C3. c-Myb mRNA expression levels in hemolytic episode patients were negatively correlated with the expression levels of miR-150. The expression of c-Myb, a regulatory factor of B lymphocytes, is increased in B lymphocytes of AIHA/Evans patients, while miR-150 expression is decreased. c-Myb was negatively correlated with miR-150.

Stability of Syk protein and mRNA in human peripheral blood basophils.

In human basophils, Syk expression is 10-fold lower than most other types of leukocytes. There are indirect studies that suggest that Syk protein is highly unstable (a calculated half-life less than 15 min) in human peripheral blood basophils. Therefore, in these studies, Syk stability was directly examined. Purified basophils were metabolically labeled and a pulse-chase experimental design showed Syk protein to be stable in the time frame of 12 h (95% likelihood that half-life is more than 12 h). However, its synthetic rate was very slow (∼10-fold slower) compared with CD34-derived basophils, which have been shown to express levels of Syk consistent with other mature circulating leukocytes. Syk mRNA expression was found to be 5-30-fold lower than other cell types (CD34-derived basophils, peripheral blood eosinophils, and plasmacytoid dendritic cells). Syk protein and mRNA levels, across cell types, were relatively concordant. Syk mRNA in basophils showed a half-life of 3.5 h, which was greater than that of interleukin-4 or Fc epsilon receptor I-α mRNA (∼2 h), but somewhat shorter than Fc epsilon receptor I-β mRNA (8 h). A comparison of miR expression between CD34-derived and peripheral blood basophils demonstrated only 1 significant increase, in miR-150 (77-fold). Transfection in human embryonic kidney cells of a stabilized form of miR-150 showed that it modified expression of c-Myb mRNA but not of Syk mRNA or protein. These results suggest that low Syk expression in basophils results, not from protein instability and perhaps not from mRNA stability. Instead, the results point to the transcriptional nature of an important point of regulation.

Pharmaceutical drugs supporting regeneration of small-intestinal mucosa severely damaged by ionizing radiation in mice

Accidental exposure of the abdomen to high-dose radiation leads to severe consequences initiated by disruption of the mucosa in the small intestine. Therapeutic options are limited, even though various treatments have been investigated, particularly in the field of regenerative therapy. In order to identify readily available treatment methods, we included several current pharmaceutical drugs, for which the clinical trials have already been completed, in tests on mice that had undergone severe mucosal damage by radiation. The drugs were injected into mice 24 h after exposure to 15.7 Gy X-rays. The effects of the drugs on the damaged mucosa of the small intestine were evaluated using early regeneration indices [the expression of c-myb mRNA, and proliferation of epithelial cells in the form of microcolonies (MCs) by Days 4 and 5 post-irradiation] and the survival rate of the mice. Enhancement of mucosal regeneration at Day 4 (c-myb: P < 0.01, MC: P < 0.05) and improvement of the survival rate (P < 0.05) were observed when a clinical dose of gonadotropin, a stimulator of androgen, was injected. Similarly, a clinical dose of thiamazole (which prevents secretion of thyroid hormone) stimulated mucosal growth by Day 5 (c-myb: P < 0.01, MC: P < 0.05) and also improved the survival rate (P < 0.05). The nonclinical drugs histamine and high-dose octreotide (a growth hormone antagonist) also gave significant survival-enhancing benefits (P < 0.01 and P < 0.05, respectively). These results can be used to construct therapeutic programs and applied in various experimental studies to control the regeneration of damaged mucosa.

C-erbB2andc-mybInduce Mouse Oocyte Maturation Involving Activation of Maturation Promoting Factor

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.

Acceleration of Regeneration of Mucosa in Small Intestine Damaged by Ionizing Radiation Using Anabolic Steroids

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells

AbstractThe c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)–15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3′-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G1 phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3′-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34+ cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb–miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.

The c-myb Protooncogene and microRNA (miR)-15a Form an Autoregulatory Loop That May Play a Role in Normal Human Erythropoiesis.

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor that contributes to lineage commitment, proliferation, and differentiation. Factors which regulate c-myb expression are of interest but remain incompletely defined. MicroRNAs (miRNAs) are being increasingly recognized as important regulators of cell development, and abnormalities in miRNA activity may also contribute to the pathogenesis of several hematologic malignancies. We speculated that miRNAs might also regulate c-Myb expression, a gene often aberrantly expressed in leukemia, lymphoma, and myeloma. Accordingly, we searched for potential miRNA binding sites in the 3′-UTR of the c-myb mRNA using the TargetScanS Target Database and identified 14 candidate miRNAs. Based on binding probability, three miRNAs (miR-15a, −107 and −150) were selected for further analysis using a luciferase reporter assay. 1191 bp of the human c-Myb 3′-UTR was subcloned downstream of the f-luc open reading frame to create the reporter construct (pBub1/Myb3U). This was co-transfected into HEK293T cell line with pRL-CMV (to normalize for transfection differences) and then either a control RNA oligonucleotide (ON) (miR-Control), miR-15a, miR-107, or miR-150 ONs. Relative luciferase activity of the pBub1/Myb3U construct was markedly diminished in cells co-transfected with miR-15a (74.1 ± 1.5%) or miR-107 (68.2 ± 5.3%) ONs, but only modestly with miR-150 ON (22.1 ± 4.2%). Functionality of miR-15a site was further tested by mutating the predicted miR-15a binding sites. This resulted in a 2 to 3 fold increase in luciferase activity, suggesting that miR-15a bound the predicated sites, and that they might be physiologically relevant. To test this possibility, miR-15a ONs were transfected into K562 human myeloid leukemia cells and the effects on c-Myb mRNA and MYB protein levels were determined. As expected with functional miRNA, c-myb mRNA levels did not change when compared to control treated cells, as measured by quantitative real-time PCR, but Myb protein levels were significantly decreased. Additionally, when analyzed by flow cytometry, miR-15a transfected cells were found to be arrested in G1 as might be expected in a Myb knockdown experiment. Conversely, when K562 cells were transfected with a methylphosphonate inhibitor (antisense) of miR-15a, endogenous c-myb expression increased. Moreover, exogenous expression in K562 cells of a c-Myb mRNA construct devoid of its 3′-UTR partially rescued the miR-15a induced cell cycle arrest. Interestingly, miR-15a levels were found to vary inversely with c-Myb mRNA expression levels in normal human CD34+ cells stimulated to develop along the erythroid, but not the myeloid lineage. These results suggested that miR-15a might play a specific, and potentially important role in regulating normal human erythropoiesis by modulating the expression of c-Myb, though we cannot exclude the possibility that additional miR-15a targets are also important. Finally, a siRNA mediated knockdown of c-Myb expression in K562 cells resulted in reduction of miR-15a expression, suggesting the possibility that Myb and miR-15a autoregulate their expression through a negative feed-back loop. We conclude that miR-15a regulates c-Myb expression in a physiologically significant, lineage specific manner in normal human hematopoietic cells. Since miR-15a localizes to ch13q14, a region often deleted in hematologic malignancies, we postulate that it might also play a role in leukemogenesis. This possibility is under active investigation in our laboratory.

α7 Nicotinic Receptor Gene Promoter Polymorphisms in Inbred Mice Affect Expression in a Cell Type-specific Fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

Identification of clinically-informative biomarkers for risk stratification within the spectrum of gastro-esophageal reflux disease in the South African population

Background : Barrett's esophagus is the precursor of esophageal adenocarcinoma with a 5-year survival rate of 25-30%. Objective : To define clinically useful biomarkers for transcriptional profiling in South African patients with Barrett's esophagus in order to identify those patients with an increased cancer risk necessitating intensified surveillance intervals. Materials and method : One-hundred and two patients were recruited for the study. COX-2, c-myb and c-myc mRNA expression were measured using a quantitative PCR method in endoscopically obtained specimens of Barrett's metaplasia (BM, n=26) or dysplasia (BD, n=14) and matching squamous esophageal tissue (n=40), squamous esophageal tissue of patients with erosive esophagitis (n=20), non-erosive esophagitis (n=20) and normal controls (n=20). Two patients with Barrett's adenocarcinoma (BAC) were also studied. Results : Demographic data of the groups were comparable. No significant differences in m-RNA expression levels were observed between ethnic groups for the genes analyzed. In the BD/BAC group 69% (11/16) showed increased c-myb m-RNA expression compared with 35% (9/26) in the BM group (p = 0.03). In the BD patients 19% (3/16) had increased c-myc m-RNA expression compared to none in those with BM and BAC. One patient each with BM and BAC had increased COX-2 m-RNA levels. No significant associations were observed for COX-2 in any of the other study groups. Conclusion : In the South African study cohort c-myb appears to be a clinically useful molecular marker for Barrett's esophagus and increased cancer risk, since m-RNA levels are progressively more over expressed in the metaplasia-dysplasia-adenocarcinoma sequence. Further studies are required to clarify the potential significance c-myc and COX-2 in South African patients with Barrett's esophagus. South African Gastroenterology Review Vol. 3(1) 2005: 5-9

2'-Deoxy-2'-fluoro- -D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2′F-ANA–DNA chimeras (PS-2′FANA–DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a >90% knockdown of c-MYB mRNA and protein expression, but the PS-2′F-ANA–DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2′F-ANA–DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.

A Novel 2′-Deoxy-2′-Fluoro (2′F-ANA) Ribose Modification Significantly Enhances the Duration, and Efficiency, of Nucleic Acid Mediated Gene Silencing.

The field of nucleic acid mediated gene silencing has been reinvigorated with the widespread adoption of RNA interference using siRNA. Since issues of delivery, stability, and duration of effect remain relevant to siRNA, and all other types of gene silencing nucleic acids, we are studying chemical modifications that may enhance the efficiency of molecules employed for this purpose. To this end, we synthesized 2′-deoxy-2′-fluoro-d-arabinonucleic acid (2′F-ANA) modifications of DNA as our prior work suggested that they would simultaneously raise the Tm of mRNA:DNA hybrids, increase resistance to nucleases, and very importantly, still permit RNaseH binding and catalysis of the target mRNA because the 2′F-ANA of the arabinose sugar ring projects, like DNA, into the major groove of the helix. Accordingly, we compared the gene silencing efficacy of 2′F-ANA modified oligonucleotides (ON) with traditional phosphorothioate (PS) antisense oligodeoxynucleotides (AS ODN) with respect to cellular delivery, intracellular stability, dose response, and duration of gene silencing in living myeloid leukemia cells. 2′F-ANA modification were made to form either “altimers” where triplets of nucleotides with F-ANA modified sugars alternated with triplets of nucleotides with deoxyribose sugars, or “gapmers” where 7, 2′fluorinated oligonucleotides flank 7 central unmodified nucleotides. The mRNA target for these comparative studies was a region within the c-myb mRNA that was previously shown by us to be accessible for hybridization in vivo (Nucleic Acids Res. 32:5791, 2004). The nucleic acids were delivered by nucleofection into K562 cells. When analyzed at 24 hours, the PS ODN and 2′F-ANA ONs demonstrated an equivalent ability to silence c-myb mRNA and protein expression (>90% compared to untreated controls). Of significant interest however, the silencing effect of the 2′F-ANA ONs was still demonstrable 96 hours post nucleoporation whereas the PS ODN lost activity after 48 hours. Further, at doses where the traditional PS ODN had no silencing effect on c-myb mRNA or protein expression (1 μg/106 cells), the 2′F-ANA ON still gave >80% suppression of the target mRNA and protein. These effects were not dependent on delivery, which appeared to be equivalent for the two chemistries. Rather, when intracellular levels of delivered material were measured by semi-quantitative slot blotting, it was shown that intracellular levels of PS ODN declined rapidly after 24 hours, whereas ~90% of the 2′F-ANA introduced into the cells was still detectable 96 hours post nucleoporation. Whether this is due to relative inability to export the 2′F-ANA out of the cell, and or diminished intracellular degradation is being investigated. Although our primary results suggested that PS ODN are more rapidly degradated in cell lysate compared to 2′F-ANA ON. Therefore, our data suggest that 2′F-ANA AS ODN are efficient gene silencing molecules with advantages over PS ODN. These include significantly greater potency, and a duration of effect that is 3-4 times longer after a single dose. These findings suggest that appropriately targeted 2′F-ANA ON, in the form of single stranded antisense molecules, or siRNA, could well prove therapeutically useful for the treatment of cancer, and in other diseases where gene silencing is expected to beneficial.

Antisense Oligodeoxynucleotide Directed against c-myb Has Anticancer Activity and Potentiates the Antiproliferative Effect of Conventional Anticancer Drugs Acting by Different Mechanisms in Human Colorectal Cancer Cells

The c-myb proto-oncogene encodes a DNA-binding protein with transactivation properties that plays an important regulatory role in cell proliferation and differentiation. Overexpression of c-myb in colonic tumors compared to normal mucosa suggests that c-myb may play a role in the malignant transformation of colonic mucosa and that inhibition of c-myb expression may suppress, to some extent, the proliferation of neoplastic cells. Complete suppression of tumor cell proliferation may require inhibition of multiple growth-promoting genes. Alternatively, the combination of oncogene-targeted oligodeoxynucleotides (ODNs) with standard cytotoxic agents might represent a useful therapeutic approach to improving cancer treatment. In the present study, we have investigated whether the inhibition of a growth-promoting gene, namely c-myb, affects the sensitivity of human colorectal cancer cells, in vitro, to conventional chemotherapeutic drugs: taxol, 5-fluorouracil, vinblastine and doxorubicin. We show that c-myb antisense phosphorothioate (S) ODN treatment induces growth arrest in the G₁/G₂ phases of the cell cycle and inhibits cell proliferation in a dose- and time-dependent manner. Also, treatment with c-myb antisense (S)ODN decreases c-myb mRNA and protein expression. A greater inhibition of cell proliferation in vitro was obtained with the combination of c-myb (S)ODN and cytotoxic drugs. The combinations exerted additive and synergistic effects on human colorectal cancer cells. This study suggests that c-myb antisense (S)ODN might be useful in the therapy of colorectal cancer in combination with chemotherapeutic drugs.

Increased c-myb mRNA Expression in Barrett's Esophagus and Barrett's-Associated Adenocarcinoma

Background. Esophageal adenocarcinoma develops through a multistage process which is characterized histopathologically by progression from Barrett's intestinal metaplasia to Barrett's esophagus with dysplasia and ultimately to adenocarcinoma. The genetic basis of this process is increasingly well understood, but no studies have examined the role of the transcription factor c-myb in this disease.Material and methods. c-myb mRNA expression levels were measured using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method in specimens of Barrett's intestinal metaplasia (n = 16), adenocarcinoma (n = 22), matching normal squamous esophagus tissues (n = 38), and normal squamous esophagus tissues from patients without Barrett's esophagus or chronic gastroesophageal reflux disease (n = 10).Results. The median c-myb mRNA expression levels were significantly increased in Barrett's intestinal metaplasia tissues compared to normal esophagus tissues (P = 0.013) and in Barrett's-associated adenocarcinoma tissues compared to normal squamous esophagus tissues (P = 0.001). The c-myb expression levels increased progressively and significantly in histopathologically worse tissue types, with an increase from normal squamous esophagus mucosa to Barrett's intestinal metaplasia, and from Barrett's intestinal metaplasia to adenocarcinoma of the esophagus (P = 0.002). Median c-myb expression levels were also significantly higher in histologically normal squamous esophagus tissues from cancer patients compared to normal esophagus tissues from patients without cancer (P < 0.001) and a control group without evidence of Barrett's esophagus or gastroesophageal reflux disease (P = 0.003). Very high c-myb mRNA expression levels were found only in patients with cancer.Conclusion. These findings suggest that upregulation of c-myb mRNA expression is an early event in the development of Barrett's esophagus and associated adenocarcinoma, that high c-myb mRNA expression levels may be a clinically useful biomarker for the detection of occult adenocarcinoma, and that a widespread cancer “field” effect is present in the esophagus of patients with Barrett's-associated adenocarcinoma.

Constitutive expression of c-myb mRNA in the adult rat brain

In this study, we demonstrated the c-myb mRNA expression in the adult rat brain using an in situ hybridization technique. We found c-myb mRNA signals in the various regions of the forebrain and midbrain including the cerebral cortex, thalamus, hippocampus, hypothalamus, superior and inferior colliculi and central gray. In the cerebellum, a diffuse signal was found in the granular layer while some positive cells were detected in the molecular layer as well. In addition, a number of cells showed intense signals in many nuclei of the medulla oblongata. The constitutive expression of c-myb mRNA in the different kinds of neural cells suggests that this gene might be involved in the normal function of these neurons.

Upregulation of c-myb mRNA expression in barrett's esophagus

Upregulation of c-myb mRNA expression in barrett's esophagus

Intron 1 rather than 5′ flanking sequence mediates cell type-specific expression of c-myb at level of transcription elongation

Previous studies have shown that expression of steady-state c-myb mRNA was regulated primarily by a block in intron 1 during transcription elongation. This study shows that the block site maps approximately 1700 bp from the start of the intron. Studies based on a reporter construct containing c-myb flanking region and intron 1 suggest that the flanking region is not important in the regulation of the cell type-specific expression of c-myb. RNA splicing of intron 1 may enhance the expression in a non-cell type-specific manner. A conserved intron domain comprising the block site is required for defining this site, but this function of the domain is independent of cell type. The cell type-specific regulation of c-myb transcription elongation is mediated by a 5′ intron sequence. A mechanism for down regulation of c-myb gene expression by the block to transcription elongation has been proposed.

Bcl-3 Expression and Nuclear Translocation Are Induced by Granulocyte-Macrophage Colony-Stimulating Factor and Erythropoietin in Proliferating Human Erythroid Precursors

AbstractBcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IκB family of proteins. In this report, involvement of Bcl-3in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)–derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation ofBcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-κB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus–type 1 (HIV-1) κB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of κB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a κB enhancer in the c-mybpromoter together with NF-κB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of ac-myb κB-TATA-luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain κB-containing genes involved in hematopoiesis, includingc-myb.© 1998 by The American Society of Hematology.

Bcl-3 Expression and Nuclear Translocation Are Induced by Granulocyte-Macrophage Colony-Stimulating Factor and Erythropoietin in Proliferating Human Erythroid Precursors

Bcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IkappaB family of proteins. In this report, involvement of Bcl-3 in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)-derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation of Bcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-kappaB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus-type 1 (HIV-1) kappaB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of kappaB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a kappaB enhancer in the c-myb promoter together with NF-kappaB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of a c-myb kappaB-TATA-luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain kappaB-containing genes involved in hematopoiesis, including c-myb.

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.

A relation between c-myc expression and prognosis of leukemia patients in Indonesia.

Overexpression of c-myc, c-myb and c-fos proto-oncogene has been observed in many leukemia cells. However, a relation between those oncogene expressions and prognosis of the leukemia is not known in Indonesia. In order to elucidate the relation, expression of those oncogenes in leukemia cells from 52 patients in Indonesia was examined by Northern blot analysis and was compared with prognosis of the disease. The leukemia patients who survived for more than 2 years were 37% of the 52 patients. Many of the samples expressed c-myc mRNA (92%). Although strong expression of c-myb and c-fos mRNA was detected in the samples (c-myb expression in 35% of the leukemia cells and c-fos in 52%), those co-expressions with c-myc mRNA did not alter the prognosis. On the contrary, all of the 4 patients suffering from leukemia which did not express c-myc mRNA survived for more than 2 years. Therefore, c-myc expression in leukemia cells may be used as an indicator for deciding prognosis of the leukemia patients in Indonesia.