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Objective To investigate the mineralization impact of fibroblast growth factor receptors 1 dominant negative (FGFR1DN) on osteogenic induction culture of bone marrow stromal cells (BMSCs).Methods The 3rd generation BMSCs were divided into 4 equal groups (n =24).FGFR1-DN group was transfected by pcDNA3.1 (+)-FGFR1DN,FGFRI group by pcDNA3.1 (+)-FRFR1,blank load group by pcDNA3.1 (+)-blank vehicle and non-transtection group by nothing.After successful transfection was confirmed when the cells were in the logarithmic phase,osteogenic induction culture was conducted continuously for 21 days.Mineralized nodule formation was observed by alizarin red staining.The amount of mineralized material was calculated according to the standard curve of alizarin red concentration.Results Continuous osteogenic induction for 21 days showed on the bottom of the hole visible round opaque calcified nodules after alizarin red staining.The BMSCs in the FGFR1-DN group induced in the logarithmic growth phase by osteoblasts exhibited significantly increased osteogenic capacity while those in the FGFR1 group displayed diminished osteogenic capacity.The concentration of alizarin red was the highest in the FGFRI-DN group (1.33 ±0.19),the lowest in the FGFRI group (1.00 ± 1.17),and moderate in the blank load group (1.20 ± 0.16) and non-transfection group (1.17 ± 0.17),showing significant between-group differences (P ＜ O.05).Conclusions FGFR1-DN can promote cell proliferation in the early differentiation of BMSCs and also mineralization of osteoblasts in bone induction culture after the logarithmic growth phase.This may provide a hint for local gene treatment of bone defects.
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Objective To investigate the mineralization impact of fibroblast growth factor receptors 1 dominant negative (FGFR1DN) on osteogenic induction culture of bone marrow stromal cells (BMSCs).Methods The 3rd generation BMSCs were divided into 4 equal groups (n =24).FGFR1-DN group was transfected by pcDNA3.1 (+)-FGFR1DN,FGFRI group by pcDNA3.1 (+)-FRFR1,blank load group by pcDNA3.1 (+)-blank vehicle and non-transtection group by nothing.After successful transfection was confirmed when the cells were in the logarithmic phase,osteogenic induction culture was conducted continuously for 21 days.Mineralized nodule formation was observed by alizarin red staining.The amount of mineralized material was calculated according to the standard curve of alizarin red concentration.Results Continuous osteogenic induction for 21 days showed on the bottom of the hole visible round opaque calcified nodules after alizarin red staining.The BMSCs in the FGFR1-DN group induced in the logarithmic growth phase by osteoblasts exhibited significantly increased osteogenic capacity while those in the FGFR1 group displayed diminished osteogenic capacity.The concentration of alizarin red was the highest in the FGFRI-DN group (1.33 ±0.19),the lowest in the FGFRI group (1.00 ± 1.17),and moderate in the blank load group (1.20 ± 0.16) and non-transfection group (1.17 ± 0.17),showing significant between-group differences (P ＜ O.05).Conclusions FGFR1-DN can promote cell proliferation in the early differentiation of BMSCs and also mineralization of osteoblasts in bone induction culture after the logarithmic growth phase.This may provide a hint for local gene treatment of bone defects.
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Objective To explore the effect of fibroblast growth factor receptors 1-dominant negative strategy (FGFR1-DN) on alkaline phosphatase (ALP) activity of bone marrow stromal stem cells (BMSCs) after osteogenic induction.Methods BMSCs were transfected with eukaryotic expression plasmid pcDNA 3.1 (+)-DN FGFR1 and pcDNA3.1 (+)-FGFR1.The experiment was conducted in 4 groups:FGFR1-DN transfection group,FGFR1 transfection group,pcDNA3.1(+) empty vector transfection group and non-transfection group.The ALP activity of BMSCs was detected in logarithmic growth phase after osteogenic culture.The qualitative detection of ALP activity was carried out immunohistochemically while the quantitative detection by cALP kit.The ALP activity was compared between the 4 groups at 7 and 14 days after osteogenic induction.Results Compared with 7 days,the ALP activity at 14 days was significantly increased in the 4 groups,and the increase in FGFR1-DN transfection group was significantly higher than in the other 3 groups (P ＜ 0.05).At both 7 and 14 days,the ALP activity in FGFR1-DN transfection group was the highest while that in FGFR1 transfection group was the lowest (P ＜ 0.05).Conclusions FGFR1-DN can promote the ALP activity of BMSCs during osteogenesis.This may provide an experimental basis for the joint application of local gene therapy and tissue engineering and for construction of tissue engineered bone with better biocompatibility.
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The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1–204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1–204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins.
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Ikaros is a transcriptional factor playing an essential role in lymphoid lineage development and differentiation. Ikaros gene deletions occur in some acute lymphoblastic leukemia (ALL) patients, and the most common type of abnormality is overexpression of dominant negative isoforms 6 (Ik6). Deletion of Ikaros gene has an independent association with a very poor outcome in B-cell-progenitor ALL. A new subtype of ALL characterized by the deletion of Ikaros and poor outcome has been identified by researchers, and named BCR/ABL1 like ALL.We can conclude that Ikaros may play an important role in the diagnosis and treatment of pediatric ALL.
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A síndrome de resistência ao hormônio tireoidiano é doença genética, caracterizada pela resposta reduzida dos tecidos-alvo ao hormônio tireoidiano. É autossômica dominante, causada, na maioria das vezes, por mutações na isoforma beta do receptor de hormônio tireoidiano (TR?). Os pacientes com a síndrome apresentam níveis séricos elevados de tiroxina livre (T4) e tri-iodotironina livre (T3) associados a feedback negativo anormal na regulação do hormônio estimulador da tireoide (TSH) e do hormônio liberador de TSH (TRH). Por essa razão, os níveis do hormônioestimulador estão pouco aumentados ou inapropriadamente normais. O diagnóstico final é feito pelo sequenciamento do gene da isoforma beta do receptor de hormônio tireoidiano. Os indivíduos afetados são, na maioriadas vezes, heterozigotos com mutações no gene TR?. Na fisiopatogenia da doença, tem sido descrito que o gene mutado inibe a função do gene normal, em um fenômeno conhecido como dominância negativa que, por sua vez, ocorre nos genes regulados negativamente e positivamente pelo hormônio tireoidiano. O mecanismo molecular da dominância negativa envolve: (1) competição da ligação do receptor mutante ao DNA, (2) dimerização do receptor mutante com o receptor do retinoide X ou com o receptor normal e (3) interação do receptor mutado com correguladores,o que acarreta em falha da regulação transcricional pelo hormônio tireoidiano. A intensidade da síndrome de resistência ao hormônio tireoidiano depende, então, em parte, do nível de expressão do receptor mutado e do tipo de mutação.
Thyroid resistance hormone syndrome is a genetic disease characterized by a reduced responsiveness of target tissues to thyroid hormone. Such patients show persistent elevation of circulating free thyroxine (T4) and free triiodothyronine (T3) levels associated with nonsuppressed serum thyrotropin (TSH). Inheritance is autosomal dominant and mutations inthe TR? gene have been identified in the majority patients with the syndrome. Most patients are heterozygous, with only one mutated TR? gene. In the pathogenesis of the disease, it has been described that the TR? mutant impairs the normal function of TR wild type (TRwt), a phenomenon described as a dominant-negative effect, which occurs in positive and negative TH regulated genes. The mutant receptor diminishes TRwt function by competing with normal receptor for DNA binding, by dimerizing with retinoid X receptor or normal receptor, and also by interacting with coregulators and disrupting the transcription activity modulated by thyroid hormone.
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In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P＜0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P＜0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.
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PURPOSE: Ataxia telangiectasia mutated(ATM) is involved in DNA damage responses at different cell cycle checkpoints, and signalling pathways associated with regulation of apoptosis in response to ionizing radiation(IR). However, the signaling pathway that underlies IR-induced apoptosis in ATM cells has remained unknown. The purpose of this study was, therefore, to investigate the apoptotic pathway that underlies IR-induced apoptosis in a CT-26 cells expressing dominant negative ATM (DN-ATM). METHODS: We generated a replication-deficient recombinant adenovirus encoding the DN-ATM(Ad/DN-ATM) or control adenovirus encoding no transgene(Ad/GFP) and infected adenovirus to CT-26 cells. After infection, we examined apoptosis and apoptotic pathway by [3H]-thymidine assay, DNA fragmentation, and Western immunoblot analysis. RESULTS: DN-ATM gene served as the creation of AT phenotype in a CT-26 cells as revealed by decreased cell proliferations following IR. In addition, IR-induced apoptosis was regulated through the reduced levels of the anti-apoptotic protein Bcl-2, the increased levels of the apoptotic protein Bax, and the activation of caspase-9, caspase-3, and PARP. CONCLUSION: These results indicate that the pathway of IR-induced apoptosis in CT-26 cells expressing DN-ATM is mediated by mitochondrial signaling pathway involving the activation of caspase 9, caspase 3, and PARP.
Subject(s)
Adenoviridae , Apoptosis , Ataxia Telangiectasia , Blotting, Western , Caspase 3 , Caspase 9 , Cell Cycle Checkpoints , Colon , Colonic Neoplasms , DNA Damage , DNA Fragmentation , Phenotype , Radiation, IonizingABSTRACT
BACKGROUND: Peroxidases (Prx) of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol respectively. Hydrogen peroxide is implicated as an intracellular messenger in various cellular responses such as proliferation and differentiation. And Prx I activity is regulated by Cdc-2 mediated phosphorylation. This work was undertaken to investigate the proliferation role of peroxiredoxin III as a member of Prx family in Prx III overexpressed HeLa cell line. METHODS: To provide further evidence of proliferation, we selected Prx III stably expressed HeLa Tet-off cell lines. Cell proliferation was examined by using proliferation reagent WST-1 in the presence or absence of doxycycline. Prx III, 2-cys Prx enzymes exist as homodimer. The activation of Prx III heterodimer with induced and endogenous Prx III was examined by immunoprecipitation. RESULTS: Immunoprecipitation analysis of the induced and endogenous Prx III with anti-myc showed that the induced wild type (WT) and dominant negative (DN) Prx III from HeLa Prx III Tet-off stable cell heterodimerized with endogenous Prx III each other. And the expression level of induced Prx III was examined after addition of doxycycline. By 72 hr, the expression level of induced Prx III was diminished gradually and the half-life of the induced wild type Prx III was approximately 17 hr. The proliferation experiment demonstrated that the relative proliferation value of induced and endogenous WT Prx III stable cell has no changes but the DN Prx III induced HeLa Tet-off stable cells were lower than endogenous Prx III. CONCLUSION: In conclusion, the HeLa dominant negative Prx III Tet-off stable cells were decreased the proliferation.
Subject(s)
Humans , Cell Line , Cell Proliferation , Doxycycline , Half-Life , HeLa Cells , Hydrogen Peroxide , Immunoprecipitation , Peroxidases , Peroxiredoxin III , Peroxiredoxins , PhosphorylationABSTRACT
Objective:To explore inhibition of telomerase activity and malignant phenotype on breast cancer cells MCF7 by Dominant Negative human telomerase reverase transcriptase gene(DN-hTERT).Methods:DN-hTERT eukaryotic expression vector DN-hTERT-IRES2-EGFP and empty vector(I) IRES2-EGFP were transfected into MCF7 by lipofectamine2000,after being selected by G418,positive clones were obtained;Tthe transfected cells growth was observed with inverted fluorescence microscope.The expression levels of hTERT mRNA of transfected cells were determined by reverse transcriptase polymerase chain reaction(RT-PCR).Telomerase activity of transfected cells was measured by TRAP-ELISE.Results:The proliferation of MCF7/DN cells were inhibited by DN-hTERT transtection;The expression levels of hTERT mRNA increased in MCF7/DN.Telomeric length was shorter in MCF7/DN than that in MCF7/I.The telomerase activity measured by TRAP-ELISE was 2.36?0.12 in MCF7/DN and was 3.32?0.14 in vacant vector MCF7/I.The telomerase activity in MCF7/DN was significantly lower than in vacant vector transfected MCF7/I(P
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Objective To study the influence of transferring a dominant-negative Stat3 gene, Stat3?on colon cancer cells' proliferation and apoptosis in vitro. Methods Cell culture of human colon cancer cell line SW480 and transient transfection were used to evaluate the effect of transferring Stat3?to cancer cells. Cell proliferation, cell cycle and apoptosis were quantified by MTT and flow cytometry, respectively. The mRNA expression of Stat3's target gene cyclin D1 and bcl-xL was detected by reverse transcription polymerase chain reaction. Independent t tests were used for data statistics. Results 36 h after Stat3?plasmids transfection, proliferation of SW480 cells was significantly inhibited (t =5. 216,P = 0.006); cell proportion of G0/G1 phase increased from 40.37% to 67.25% and early apoptosis cells increased from 5. 34% to 24. 42% ; mRNA expression of cyclin Dl and bcl-xL declined significantly (t = 5.288,P=0.010;t=3.517,P=0.025). Conclusion Blocking Stat3 signaling pathway by transfection of Stat3?plasmid inhibits the proliferation and promotes apoptosis of colon cancer cells, which provides a experimental foundation of Stat3 targeted colon cancer gene therapy.
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Objective The newly developing gene therapy method and dominant negative mutants were bein g used as new promising HBV therapy method, and a dominant negative mutant of HB V X g ene we have reported in our previous report has some effects both on HBV replica tion and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG 2 2.2.15 cell line in the experiment. To mak e sure the effects of dominant negative mutant of HBx gene, we established a HBx DN stably expressing cell clone, and evaluated the effects of HBx dominant negat ive mutant on HBV gene expression. Methods The prev HBx-GFP dominant mutant and the plasmids pRev Xwt, pRev GFP which contain the wild type X gene or GFP gene then transfected into HepG 2 2.2.15 cells by liposome. The HBsAg, HBeAg by in media were as sayed by RIA and HBV-related RNA were assayed by Northern blot. Results The pRev HBx-GFP, GFP and wild type X constructs can be effectively expressed in HepG 2 2.2.15 cells. The stable expressed HBx -GFP can significantly reduce HBeAg, HBeAg in media and the HBV-related RNA in HepG 2 2.2.15 cells, but not for pRev Xwt and pRev GFP. Conclusions The dominant negative mutant pRev HBx-GFP can significantly inhibit the HBV gen e expression. It also suggested that X gene may be one promising target for HBV gene therapy.
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Objective To observe a recombinant mutant of HBV core protein for dominant negative gene therapy against HBV encapsidation in vitro. Methods C gene and S gene of HBV were acquired through PCR and subcloned into pGEM T to construct pGEM T C and pGEM T S respectively. After digestion and ligation of these two plasmids, pGEM T CS was constructed. The cloned gene was inserted into pcDNA3.1 + to construct pcDNA3.1 + CS, which was identified by DNA sequencing. The recombinant plasmids were transformed into HepG2 cells, and screened with G418. The resistant HepG2 cell clones were chosen to test the expression of core surface protein by RT PCR, and the expressing HepG2 clones were cultured with 10% HBV DNA positive human serum for 72 hours. The intracellular HBV particles were extracted and the DNA was subjected to dot hybridization. Results The analysis showed that the HepG2 cells expressing mutant C protein had capabilities to resist HBV invasion in varied degrees. The mutant C protein had a dominant negative role in the encapsidation of HBV compared with the naive part of core protein. Conclusions The production of recombinant mutant core protein has a potential value for gene therapy against HBV infection.
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We have constructed dominant-negative retinoic acid receptors by substituting a single amino acid which has been found in a dominant-negative thyroid hormone receptor, and have expressed the dominant-negative retinoic acid receptors in the epidermis, a potential target organ of retinoic acid. The resultant transgenic mice exhibited dramatic suppression of epidermal development, demonstrating the absolute requirement of retionic acid in normal skin development. This novel method, targeted expression of the dominant-negative receptor, is theoretically applicable to any organ, thus opening the way to defining the physiological roles of retionic acid as well as other lipophilic hormones during embryogenesis as well as in adults.