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This study aims to gain insight into the DNA-specific recognition mechanism of c-Myb transcription factor during the regulation of cell early differentiation and proliferation. Therefore, we chose the chicken myeloid gene, mitochondrial import protein 1 (mim-1), as a target to study the binding specificity between potential dual-Myb-binding sites. The c-Myb-binding site in mim-1 is a pseudo-palindromic sequence AACGGTT, which contains two AACNG consensuses. Simulation studies in different biological scenarios revealed that c-Myb binding with mim-1 in the forward strand (complex F) ismore stable than that inthereverse strand (complex R). The principal component analysis (PCA) dynamics trajectory analyses suggested an opening motion of the recognition helices of R2 and R3 (R2R3), resulting in the dissociation of DNA from c-Myb in complex R at 330 K, triggered by the reduced electrostatic potential on the surface of R2R3. Furthermore, the DNA confirmation and hydrogen-bond interaction analyses indicated that the major groove width of DNA increased in complex R, which affected on the hydrogen-bond formation ability between R2R3 and DNA, and directly resulted in the dissociation of DNA from R2R3. The steered molecular dynamics (SMD) simulation studies also suggested that the electrostatic potential, major groove width, and hydrogen bonds made major contribution to the DNA-specific recognition. In vitro trials confirmed the simulation results that c-Myb specifically bound to mim-1 in the forward strand. This study indicates that the three-dimensional (3D) structure features play an important role in the DNA-specific recognition mechanism by c-Myb besides the AACNG consensuses, which is beneficial to understanding the cell early differentiation and proliferation regulated by c-Myb, as well as the prediction of novel c-Myb-binding motifs in tumorigenesis.
Subject(s)
Molecular Dynamics Simulation , Consensus , DNA , HydrogenABSTRACT
Objective To evaluate the role and mechanism of miR-150 in cardiac fibrosis after MI.Methods A rat model of MI was established by up-regulating miR-150 through overexpressing miR-150 lentivirus.Real-time PCR and Western blot were applied in detecting the expression of collagen 1 α 1 and α-SMA protein in infarction area border.Masson coloration was applied in measuring fibrosis.Cardiac fibroblasts were isolated and cultured.UTR was used to report the carrier and lentivirus.And c-Myb siRNA was used to verify the relationship between c-Myb and microRNA-150.Results In vivo,MiR-150 was down-regulated in myocardium border zone in 14 day and 28 day after infarction (P ＜ 0.001,P ＜ 0.05),and overexpressing miR-150 promoted myocardial fibrosis (P ＜ 0.001),and inhibited the expression of collagen1α 1 and α-SMA (P ＜ 0.01,P ＜ 0.05).In vitro,c-Myb was the direct target gene of miR-150,and inhibited the expression of c-Myb resulting in the down regulation of collagen1α 1 and α-SMA,suggesting that the role of miR-150 was achieved by regulating c-Myb.Conclusions MiR-150 was down-regulated in myocardium border zone,and myocardial fibrosis can be improved by targeting c-Myb.
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C-myb is a member of the myb transcription factor family and involved in cell proliferation and differentiation.It activates specific target genes to regulate specific program through certain channel,and it takes signal transduction to regulate function and expression of protein.In this paper,c-myb,by binding to Wnt signaling pathway,makes Lgr5 start to promote cell proliferation and differentiation,and it can serve as a new therapeutic target to provide the basis of early diagnosis and treatment for colorectal cancer.
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Objective To study the effects of microRNA-34a-5p on erythroid differentiation of K562 cells.Methods K562 cells were transfected with the microRNA-34a-5p mimics and antisense inhibitors specifically targeting mi-croRNA-34a-5p, respectively.The effects of over-expression or knocking-down of microRNA-34a-5p were exam-ined by Quantitative RT-PCR.Flow cytometry was performed to detect specific surface marker of erythroid cells . The benzidine staining assay was used to access the differentiation of K 562 cells.Western blot was performed to de-tect miRNA targets.Results microRNA-34a-5p was down-regulated at the early stage of K562 erythroid differenti-ation.Over-expression of microRNA-34a-5p in K562 cells attenuates erythroid differentiation , in contrast, inhibi-tion of microRNA-34a-5p accelerates erythroid pheotypes in K562 cells.c-MYB was found to be the direct target of microRNA-34 a-5 p in erythroid cells .Conclusions microRNA-34 a-5 p regulates early erythroid differentiation of K562 cells via repressing c-MYB.
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Objective To investigate the roles of aberrant expression of c-myc,c-myb,and c-erbB-2 in the development of breast cancer and its clinical significance.Methods Immunohistochimical technique (S-P method)was used to detect the expression of c-myb,c-myc and c-erbB-2 protein in 150 cases of breast cancer and 30 cases of benign proliferative lesions.heir relation to clinical pathological parameters were analyzed.Results The protein expression levels of c-myc,c-myb,and c-erbB-2 in breast cancer were higher than those in benign proliferative lesions and the difference was significant(all P ＜0.05).Expression of c-myc protein had obvious relationship to histological grade,lymph nodes metastasis and pathological types(all P ＜ 0.05).Expression of c-myb protein related significantly to histological grade (P ＜ 0.05).c-erbB-2 was associated with the tumor size,grading,PR expression (all P ＜ 0.05).The association among the three kinds of genetic proteins was observed.Conclusion Expressions of oncogenes c-myc,c-myb,and c-erbB-2 proteins are higher in breast cancer and lower in benign proliferative lesions.The difference is significant.They are related to the grading and some other prognostic factors such as tumor size,pathologic types,PR expression and lymph node metastasis.The results suggest that these oncogenes are activated in the development of breast cancer and provoke cancerous cells multiplication.Expressions of oncogenes c-myc,c-myb and c-erbB-2 mightbe considered to be the prognostic indicators of breast cancer.
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BACKGROUND AND OBJECTIVES: Although the role of c-myb in head and neck tumor has not been studied, aberrant expression of c-myb in various cancer has been demonstrated recently, suggesting that c-myb may play a role in tumorigenesis. Consequently, disrupting c-myb function might prove a possible stratergy for controlling cancer cell growth. The purpose of this study is to investigate the possibility of clinical application of adenovirus-mediated dominant negative c-myb (DN-myb) gene therapy in head and neck cancer. MATERIALS AND METHOD: All tissues were obtained from patients undergoing therapeutic operation for head and neck tumors and were assayed the expression of c-myb and bcl-2 in tumor and normal tissue by RT-PCR. We have generated a replication-deficient recombinant adenovirus encoding the dominant negative c-myb (Ad/DN-myb) or control adenovirus encoding no transgene (Ad/GFP) and infected adenovirus to SNU-1076, head and neck tumor cell line. We examined cell proliferation by 3H-thymidine assay, apoptosis by DNA fragmentation, bcl-2 expression and Akt/PKB phosphorylation by Western immunoblot, and IGF-II, VEGF mRNA expression by RT-PCR. RESULTS: c-myb expression of head and neck tumor tissues was significantly higher than that of normal tissue, indicating that these genes may play an important role in carcinogenesis of head and neck tumor. Ad/DN-myb infected SNU-1076 cells decreased cell proliferation, IGF-1I and VEGF expression, and remarkably increased their apoptosis through the down-regulation of bcl-2 expression and the inhibition of Akt/PKB pathway activation. CONCLUSION: Results obtains from these study suggest that DN-myb induced apoptosis of head and neck tumor cells and the adenovirusmediated DN-myb gene therapy may be a potential molecular therapy for the treatment of head and neck tumor.
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Cell Transformation, NeoplasticABSTRACT
Objective: To study the effect of combining bcr-abl Aspo and c-myb Aspo on K562 cells. Methods: Cellswere exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU-K562 cells were culturedin 0.8% methyleellulose. P210 was measured by flow cytometry. Cellular bcr-abl mRNA was detected by RT-PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr-abl Aspo and c-myb Aspo was 5 μmol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61.7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 μmol/L bcr-abl Aspo and 10 μmol/L c-myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosisat 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr-abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 μmol/L Aspo and 10 mol/L. Conclusion: It emerges coordination to combine bcr-abl Aspo and c-myb Aspo on K562 cells, and enhances the anti-leukemia effect.
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BACKGROUND: Aberrant expression of c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos was shown to inhibit growth of normal as well as leukemic cells. C-myb antisense oligo for inhibition of tumor cell growth was, however, not decisive enough to be an effective anti-cancer agent. Thus, we set out to devise a systematic approach to find effective target sites for c-myb antisense oligos and to compare cellular uptake of antisense oligos complexed with different liposomes. METHODS: A computer simulation program for RNA secondary structures was employed to choose 8 potential target sites free of secondary structures along the entire c-myb mRNA sequence. Linear phosphorothioate-capped antisense oligos complementary to the selected target sites were synthesized and delivered into HL-60 and K562 cancer cell lines as liposomes complexes. RESULTS: Three of the 8 target sites were found to be relatively effective for reducing c-myb message. The three oliogs, MIJ-4, -17 and -18 were able to reduce c-myb message by more than 70% and suppressed tumor cell growth by about 70%. When three different cationic liposomes were used to facilitate the cellular uptake of antisense c-myb oligos, distinct liposome formulations were found to be comparably effective for reduction of c-myb message and inhibition of tumor cell growth. CONCLUSION: These results show that simulation of RNA secondary structure can be used to search effective target sites for antisense oligos and oligo uptake can be significantly enhanced by liposomes. However, cellular uptake of antisense oligos by liposomes needs further improvement.
Subject(s)
Cell Line , Computer Simulation , Genes, myb , Liposomes , RNA , RNA, MessengerABSTRACT
PURPOSE: The c-myb protooncogene encodes MYB protein that is critical for normal and leukemic hematopoietic cell proliferation and development. It is known that c-myb plays an important role in leukemogenesis as well. Aberrant expression of c-myb is seen in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), and chronic myeloid leukemia (CML). We reasoned that down regulation of c-myb expression using synthetic antisense oligomers targeted to c-myb mRNA might prove useful antileukemic agents if leukemia cells were more dependent on c-myb function for proliferation than their normal counterparts. To investigate the applying possibility of c-myb antisense oligodeoxynucleotides as the useful antileukemic agents, we examined the cell viability, cloning efficiency and the expression of c-myb mRNA and MYB protein after the exposure of c-myb oligomers on normal bone marrow cells and leukemia cells. MATERIALS AND METHODS: We maintained in short-term suspension culture for 4 days to analyze the effect of c-myb oligomers on normal bone marrow cells and chronic myelocytic leukemia cell line K562. During suspension culture, cell counts and viability were periodically determined, and immediately seeded into duplicate methylcellulose cultures containing recombinanat human interleukin 3 and GM-CSF. Cells placed in semisolid cultures were allowed to grow for an additional 10~12 days. We counted the colonies, and then RNA and protein was extracted from cells cloned in liquified methyl- cellulose cultures. We detected the c-myb mRNA expression by RT-PCR and Southern hybridization analysis and MYB expression by Western hybridization analysis. RESULTS: c-myb sense oligomers had negligible effects on K562 cells growth in short- term suspension culture, while exposure to c-myb antisense oligomers resulted in a daily decline in cell number. In contrast, normal bone marow cell viability and numbers were unaffected by c-myb sense or antisense oligomers exposure. c-myb antisense oligodeoxy nucleotides strongly inhibited or completely abolished growth and colony formation of K562 cells. In contrast, c-myb sense oligomers did not affect. At antisense dose that inhibited leukemic cell growth, normal bone marrow cells survived. Thus, normal and leukemic cells showed the differential sensitivity to the toxic effect of c-myb antisense oligomers. RT-PCR, Southern hybridization analysis and Western hybridization analysis of c-myb antisense-treated K562 cells revealed a complete absence of c-myb mRNA expression and MYB expression. CONCLUSION: Results obtained from these studies suggest that inhibition of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular biologic approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agent.
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Humans , Bone Marrow Cells , Bone Marrow Purging , Cell Count , Cell Line , Cell Proliferation , Cell Survival , Cellulose , Clone Cells , Cloning, Organism , Down-Regulation , Granulocyte-Macrophage Colony-Stimulating Factor , Interleukin-3 , K562 Cells , Leukemia , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukemia, Myeloid, Acute , Methylcellulose , Nucleotides , Oligodeoxyribonucleotides , Precursor Cell Lymphoblastic Leukemia-Lymphoma , RNA , RNA, MessengerABSTRACT
PURPOSE: Aberrant expression of the c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos could be an effective way to abort rapid growth of leukemic cells. Developing stable antisense oligos combined with enhanced delivery into cells would be of great use in developing an effective anti-cancer molecular agent. MATERIALS AND METHODS: Selection of target sites was carried out by employing computer simulation of mRNA secondary structures. Multiple antisense oligo sequences were adjoined and AS-oligos were then covalently closed to evade exonuclease activities. C-myb antisense oligos with a novel structure were complexed with cationic liposomes and used to treat HL-60 leukemic cells. RESULTS: We developed covalently closed antisense oligos which harbor four adjoined antisense sequences. The c-myb antisense oligos were found to be exceptionally stable and effective in specifically ablating c-myb mRNA. The antisense oligos were able to inhibit growth of leukemic cell line (HL-60) by about 80%. Antisense effect was more pronounced when the cells were treated twice with the antisense oligos at lower concentrations. CONCLUSION: The novel covalently closed antisense oligo (CMAS-oligos) was found to be effective and exceptionally stable, Growth of HL-60 was significantly inhibited, showing a rational way to develop an effective molecular anti-cancer agent.
Subject(s)
Cell Line , Computer Simulation , Genes, myb , Liposomes , Oligonucleotides, Antisense , RNA, MessengerABSTRACT
Objective: To study the effect of combining bcr abl Aspo and c myb Aspo on K562 cells. Methods: Cells were exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU K562 cells were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. Cellular bcr abl mRNA was detected by RT PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr abl Aspo and c myb Aspo was 5 ?mol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61 7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 ?mol/L bcr abl Aspo and 10 ?mol/L c myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosis at 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 ?mol/L Aspo and 10 ?mol/L. Conclusion: It emerges coordination to combine bcr abl Aspo and c myb Aspo on K562 cells, and enhances the anti leukemia effect.
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The respective sense and antisense oligodeoxynucleotides and their phosphorothiods, targeting at the bcr/abl, were the hallmark gene of Chronic Myelogenous Leukemia( CML) and c-myb correlates with CML. K562 cells, derived initially from a patient of CML blast crisis, were incubated with bcr/abl antisense oligodeoxynucleotides asODN or c-myb asODN or with both asODN in combinalion. All kinds of asODN could not only significantly inhibit K562 cells survival with the highest inhibitory rate of 64.7%?3.2%, but also dramatically inhibit DNA synthesis and the highest inhibitory rate was 85. 8 %?4.1%, decrease bcr/abl mRNA level almost completely and induce apoptosis significantly. The inhibition effect of antisense oligodeoxynucleotides phosphorothiodes s-asODN was stronger than that of unmodified asODN. The inhibition of the combination of bcr/abl and c-myb asODN was stronger than that of anyone alnoe. All the inhibitions exhibited in sequence-and time-dependent manner. We concluded that bcr/abl in pathogenesis of CML not only increased CML cells proliferation rate, but also decreased CML cells apoptosis rate. c-myb may participate in CML mainly by regulating bcr/abl. Trie results indicated that the modified asODN and multiple asODN targeting several oncogenes may advance to antisense gene therapy.
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98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and ? 1-Ⅰ collagen mRNA expression were repressed significantly. CONCLUSIONS: c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and ? 1-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.
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Objective The c-myb is an important transcription factor in early haemopoietic system development and differentiation. We wish to use ES cell culture system by gene target getting cell models of c-myb+/+ and c-myb-/- ES in vitro in order to examine the detailed roles of the transcription factor c-myb in haemopoietic commitment and differentiation. Methods Haemopoietic differentiation was initiated by seeding ES cells in methylcellulose medium in the absence of LIF. The embryoid bodies of c-myb+/+ and c-myb-/- ES were analyzed by methylcellulose colony assay and real-time PCR to compare the formation of each haemopoietic system on different differentiation stage and procedure and relative gene expression. Results The formation of embryoid bodies was similar for both c-myb+/+ and c-myb-/- ES cells with the exception that the size and frequency of EBs reduced in the case of the c-myb-/- cells. The number of embryoid bodies increased more markedly in c-myb+/+ than that in c-myb-/-. Erythroid progenitors (CFU-E) were first present in c-myb+/+ group at 6 d after in vitro differentiation, and their number reached the peak at 8 d. Similar kinetics were seen for the formation of CFU-Es in c-myb-/- group, but their number was lower than that in c-myb+/+ group, and the colonies were generally small. CFU-M were first detectable at 7 d, and the peak levels were present at 7 d in c-myb+/+ group. Similar kinetics were seen for the formation of CFU-M in c-myb-/- group, but their number was lower than that in the c-myb+/+ group. CFU-GM was first detectable at 7 d, and the peak levels were present at 12 d in c-myb+/+ group. CFU-GM was not detected in c-myb-/- group. Real-time PCR analysis showed that there was no change in the gene expressions of ?-globin, zeta-globin, Lys, and C-fms in the two groups of c-myb+/+ and c-myb -/-. Conclusion Low levels of c-myb are suff-icient to allow progenitor expansion, but progression of progeniters towards terminal differentiation is significantly altered. Low levels of c-myb can influence erythropoiesis development, but precursor cells capable of differentiating into macrophages are present in haemopoietic commitment. The levels of c-myb can not change the gene expressions of ?-globin, zeta-globin, Lys, and C-fms.