Recognition and Suppression of Transfected Plasmids by Protein ZNF511-PRAP1, a Potential Molecular Barrier to Transgene Expression

Qiu, Guo-Hua; Leung, Carol Ho-Wing; Tong, Yongxi; Xie, Xiaojin; Laban, Mirtha; Hooi, Shing Chuan

doi:10.1038/mt.2011.80

Cited by 3 publications

(2 citation statements)

References 44 publications

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“…Total cellular RNA was extracted from cells cultured in six-well plates using RNeasy Kit (Qiagen), according to manufacturer’s instructions. Traditional RT-PCR ( 6 ) and Real-time RT-PCR ( 28 ) were conducted as previously described, respectively. The used primers are listed in Table 1 or as described in previous papers ( 6 , 29 ).…”

Section: Methodsmentioning

confidence: 99%

Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

et al. 2015

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Background:The molecular mechanisms of tumor suppressor gene DLEC1 are largely unknown.Objectives:In this study, we established DLEC1 over-expression stable clones to study the cellular function of DLEC1 in the colorectal cancer cell line, HCT116.Materials and Methods:Stable clones with DLEC1 over-expression were first established by the transfection of DLEC1 expression construct pcDNA31DLEC1 in HCT116. On G418 selection, positive stable clones were screened for DLEC1 expression level by conventional reverse transcription-polymerase chain reaction (RT-PCR), and verified by real-time RT-PCR and Western blotting. Subsequently, these stable clones were subjected to colony formation and cell cycle analyses and identification of factors involved in G1 arrest. Lastly, three stable clones, DLEC1-7 (highest DLEC1 expression), DLEC1-3 (lowest expression) and pcDNA31 vector control, were employed to analyze cell proliferation and cell cycle after AP-2α2 knockdown by siRNAs.Results:The DLEC1 over-expression was found to reduce the number of colonies in colony formation and to induce G1 arrest in seven clones, and apoptosis in one clone in the cell cycle analysis. Furthermore, regardless of the different cell cycle defects in all eight stable clones, the expression level of transcriptional factor AP-2α2 was found to be elevated. More interestingly, we found that when AP-2α2 was knocked down, DLEC1 over-expression neither suppressed cancer cell growth nor induced G1 arrest, yet, instead promoted cell growth and decreased cells in the G1 fraction. This promotion of cell proliferation and release of G1 cells also seemed to be proportional to DLEC1 expression levels in DLEC1 stable clones.Conclusions:DLEC1 suppresses tumor cell growth the presence of AP-2α2 and stimulates cell proliferation in the down-regulation of AP-2α2 in DLEC1 over-expression stable clones of HTC116.

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Section: Methodsmentioning

confidence: 99%

Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

et al. 2015

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“…Loss of transgene expression from episomal DNA both in vitro and in vivo is widely acknowledged. This loss is mediated through nuclease activity or remodelling of chromatin architecture (Hasse, Schulz et al 1992, Bishop, Ramalho et al 2006, Riu, Chen et al 2007) particularly within expression constructs utilising a viral promoter sequence to drive protein expression (Qiu, Leung et al 2011). Retention of episomal expression over time is highly variable between cell lines due to differences in the activity of plasmid degradation processes, along with differences in the rates of plasmid dilution through cellular proliferation.…”

Section: Rationalising the Abundance Of Tgfp-d297i-βi-tubulin Expression In Hek293 Cellsmentioning

confidence: 99%

Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

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<p>Marine environments represent a rich source of bioactive secondary metabolites that may be harnessed for use in a therapeutic context. Two novel compounds, peloruside A and laulimalide, isolated from the marine sponges Mycale hentsheli and Cacospongia mycofijiensis, respectively, both demonstrate useful pharmacological properties in mammalian cells. These compounds share major similarities with microtubule-stabilising agents. Like other agents in this class, peloruside A and laulimalide bind to the β-tubulin subunit of microtubules, the primary cytoskeletal element of eukaryotic cells. These compounds enhance polymerisation dynamics between ternary microtubule structures and severely hinder necessary cytoskeletal rearrangements within the cell. Over the course of a patient’s treatment, cancerous cells may develop multi-drug resistance phenotypes. P-glycoprotein drug efflux pumps play a major role in the development of therapy resistance in many cancers, as the current generation microtubule-stabilising agents are easily removed from diseased cells by upregulated efflux mechanisms. Unlike agents already in clinical application, both peloruside A and laulimalide are poor substrates for removal by these mechanisms, making them and their synthetic derivatives interesting as potential treatments for drug-resistant tumours. Peloruside A and laulimalide exhibit potent nanomolar anti-mitotic activities in vitro and arrest cell cycle progression in G₂/M phase, leading to cell death – a characteristic mode of action among microtubule-stabilising agents. Unlike all known agents in this class, peloruside A and laulimalide share a secondary, unique binding region in β-tubulin. In the past decade our understanding of this region has developed, revealing a second, unique mechanism for stabilisation of microtubules. Using mammalian cells to model physiological tubulin, the present study investigates the predicted role of aspartic acid 297 of human βI-tubulin in the binding association of both peloruside A and laulimalide. This particular amino acid is predicted to hydrogen bond with both compounds, contributing to their activity as stabilisers. It was revealed that the introduction of a point mutation in D297 resulted in a small but highly consistent resistance phenotype to both compounds, but not to microtubule-stabilising agents that bind to the traditional, taxoid site on β-tubulin. It was concluded that aspartic acid 297 is likely to be one of the amino acids directly involved in the binding association of peloruside A and laulimalide to β-tubulin, contributing partial compound stabilisation. The rational synthesis of future analogues may benefit from these findings in the design of molecules with enhanced interactions at this particular amino acid residue.</p>

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A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

et al. 2016

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Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson's R 2 of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.

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Recognition and Suppression of Transfected Plasmids by Protein ZNF511-PRAP1, a Potential Molecular Barrier to Transgene Expression

Cited by 3 publications

References 44 publications

Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

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