Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

Sacramento, Chester Bittencourt; Moraes, Jane Zveiter de; Denapolis, P.m.a.; Han, Sang Won

doi:10.1590/s0100-879x2010007500064

Cited by 18 publications

(12 citation statements)

References 22 publications

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“…HCT-116, LS174T and HCT8 cells were cultured in RPMI-1640 (Gibco, USA) medium with 10% fetal bovine serum (FBS) (Gibco, USA), whereas HT-29 cells were cultured in McCoy’s 5A(Gibco, USA) medium with 10% FBS. All cells were cultured with 100 μg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA)12 at 37 °C with 5% CO 2 . The EMT inhibitor U0126-EtOH(SELLECK, S1102, USA) and SB431542(SELLECK, S1067, USA).…”

Section: Methodsmentioning

confidence: 99%

Hypoxia-induced vasculogenic mimicry formation in human colorectal cancer cells: Involvement of HIF-1a, Claudin-4, and E-cadherin and Vimentin

Zong

Shi

et al. 2016

Sci Rep

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Vasculogenic mimicry (VM) plays an important role in colorectal cancer (CRC) metastasis, and both hypoxia and the epithelial-mesenchymal transition (EMT) are necessary for VM. In this study, HIF-1α expression was upregulated in the VM-positive CRC cell line HCT-116 and thereby affected the expression of the EMT-related markers Claudin-4, E-cadherin (E-cd) and Vimentin(VIM). SB431542 and U0126EtOH, which can inhibit of EMT were used to treat HCT-116 and HCT-8 in these experiments. Both of the inhibitors had significant effect on EMT markers and the formations of VM in CRC cells. In addition, knockdown of HIF-1α in the HCT-116 cells inhibited their capacity for VM. Our study reveals a regulatory role for HIF-1α in VM and suggests that targeting either HIF-1α or EMT may be a valuable strategy for the elimination of CRC metastasis.

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Section: Methodsmentioning

confidence: 99%

Hypoxia-induced vasculogenic mimicry formation in human colorectal cancer cells: Involvement of HIF-1a, Claudin-4, and E-cadherin and Vimentin

Zong

Shi

et al. 2016

Sci Rep

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“…The sequence binds to a transcription factor which then undergoes a morphological change, revealing its nuclear localization signal (NLS) [19]. The DNA and its indirectly associated NLS can then be transported into the nucleus.…”

Section: Methodsmentioning

confidence: 99%

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

et al. 2016

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Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6 ± 0.6% of the cells with 101 ± 3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24 hours post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62 hr−1 and 0.084 hr−1 for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10−4 hr−1) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1 hr−1). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles.

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“…This vector was digested with NruI and was ligated with the attB sequence from the pTA-attB vector (kindly provided by Dr Michele P. Calos, Genetics department of Stanford University, USA [20]), which was previously digested with BamHI and EcoRV and treated with Klenow polymerase, to make pVAX-HRE-attB. Finally, to insert the human VEGF165 sequence into pVAX-HRE-attB, both it and the uP-VEGF vector [23], which expresses human VEGF165, were digested with HindIII and ApaI and ligated together to make pVAX-HRE-attB-VEGF. The integrase expression vector uP-INT was constructed by inserting the ΦC31 integrase gene into the uP vector.…”

Section: Methodsmentioning

confidence: 99%

Treatment of Mouse Limb Ischemia with an Integrative Hypoxia-Responsive Vector Expressing the Vascular Endothelial Growth Factor Gene

Yasumura¹,

Stilhano²,

Samoto³

et al. 2012

PLoS ONE

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Constitutive vascular endothelial growth factor (VEGF) gene expression systems have been extensively used to treat peripheral arterial diseases, but most of the results have not been satisfactory. In this study, we designed a plasmid vector with a hypoxia-responsive element sequence incorporated into it with the phiC31 integrative system (pVHAVI) to allow long-term VEGF gene expression and to be activated under hypoxia. Repeated activations of VEGF gene expression under hypoxia were confirmed in HEK293 and C2C12 cells transfected with pVHAVI. In limb ischemic mice, the local administration of pVHAVI promoted gastrocnemius mass and force recovery and ameliorated limb necrosis much better than the group treated with hypoxia-insensitive vector, even this last group had produced more VEGF in muscle. Histological analyses carried out after four weeks of gene therapy showed increased capillary density and matured vessels, and reduced number of necrotic cells and fibrosis in pVHAVI treated group. By our study, we demonstrate that the presence of high concentration of VEGF in ischemic tissue is not beneficial or is less beneficial than maintaining a lower but sufficient and long-term concentration of VEGF locally.

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Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

Cited by 18 publications

References 22 publications

Hypoxia-induced vasculogenic mimicry formation in human colorectal cancer cells: Involvement of HIF-1a, Claudin-4, and E-cadherin and Vimentin

Hypoxia-induced vasculogenic mimicry formation in human colorectal cancer cells: Involvement of HIF-1a, Claudin-4, and E-cadherin and Vimentin

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

Treatment of Mouse Limb Ischemia with an Integrative Hypoxia-Responsive Vector Expressing the Vascular Endothelial Growth Factor Gene

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