Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions

Abstract

ObjectiveHIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo.MethodsHuman colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE−). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model.Results(1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6–12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging.ConclusionThe results of this study will help in the understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

“…Fomicheva et al demonstrated that in the double oxygen-sensing vector system (DOSVS), 6 HREs elicited a 3-fold induction increase compared to 2 HREs, despite an obvious increase in normoxic leakage [77]. A study by Takeuchi et al, which investigated the expression rates of luciferase reporters under the control of varying numbers of VEGF gene-derived HREs (from 2 to 12) depending on different hypoxic conditions (O 2 concentrations from 1% to 16%) and different hypoxia durations (from 6 to 24 h), was in agreement with the general trends [78]. Some studies reveal more complicated patterns of HRE copy number influence, indicating that there might be other factors that affect HIF-mediated transcription, such as the three-dimensional alignment of HREs on DNA.…”

“…It is worth mentioning that HREs act in a bidirectional way; they are capable of endowing oxygen sensitivity to 2 different genes located along their edges. Therefore, it is possible to control luciferase and β-galactosidase [62] or a double luciferase system [78] with a single enhancer element if needed.…”

Human relaxin gene expression delivered by bioreducible dendrimer polymer for post-infarct cardiac remodeling in rats

“…The loss of the transcriptional HRE in the vascular endothelial growth factor (VEGF) promoter reported adult-onset progressive motor neuron degeneration in mice with neuropathological features reminiscent of amyotrophic lateral sclerosis in humans [57]. The multicopy of HRE has been considered as a potential candidates for diagnostic and therapeutic applications [62,63]. In human colon carcinoma HCT116 cells, gene activation levels mediated by HRE showed a linear correlation with an increase of HRE copy number and a saturation effect with more than 6 or 8 copies of an HRE [63].…”

“…The multicopy of HRE has been considered as a potential candidates for diagnostic and therapeutic applications [62,63]. In human colon carcinoma HCT116 cells, gene activation levels mediated by HRE showed a linear correlation with an increase of HRE copy number and a saturation effect with more than 6 or 8 copies of an HRE [63]. However, the optimal HRE copy number for therapeutic genes was not determined in the diverse diseases yet [58].…”

Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy

“…For instance, replacing the minimal thymidine kinase (TK) promoter with the minimal SV40 promoter results in an increased induction ratio of 18-fold to 146-fold [ 35 , 45 ]. HRE copy number: Increasing the HRE copy number is a preferable option to promote enhanced gene expression [ 4 – 6 , 36 , 46 , 47 ]. Multimerisation of the HRE core sequence in different gene, such as Epo/VEGF-A , results in increase in the basal gene expression under hypoxia when compared to the aerobic controls, with a threshold of a minimum three copies.…”