Antisense DNAs as multisite genomic modulators identified by DNA microarray

Cho, Yee Sook; Kim, Meyoung Kon; Cheadle, Chris; Neary, Catherine; Becker, Kevin G.; Cho‐Chung, Yoon S.

doi:10.1073/pnas.171314398

Cited by 74 publications

(73 citation statements)

References 29 publications

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“…26,27 Moreover, microarray technology has been minimally exploited with respect to the study of adenovirus-host cell interaction. [10][11][12][13][14] Therefore, further investigations of adenoviral infection may reveal unique changes in gene expression that induced at different points in its infection pathway. In this regard, we are utilizing heat-inactivated adenoviral vectors that permit binding to the cell surface receptor but do not allow internalization of the virus, 28 to study the effects of adenoviral cell surface binding on gene expression.…”

Section: Resultsmentioning

confidence: 99%

“…Two extremely powerful and versatile technologies utilized for this purpose include high-density oligonucleotide and cDNA microarrays. The use of microarray technology has been minimally exploited in the field of gene therapy [10][11][12][13][14] particularly with respect to adenoviral 15,16 vectors and their tropismmodified variants. The intent of our study was to determine differential gene expression patterns linked to a defined set of tropism-modified adenoviral vectors.…”

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confidence: 99%

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Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

et al. 2004

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In this study, we have applied high-density oligonucleotide microarray technology to characterize biologic changes associated with adenoviral vector-mediated target cell infection. We infected a human melanoma cell line, M21, with the tropism-modified vectors, Ad5lucRGD and Ad5/3luc1. In addition, we infected the M21 cell line with the Ad5luc1, a vector which primarily exploits the coxsackie and adenovirus receptor, as its primary native receptor. We found significant changes in gene expression of 5492 genes induced by Ad5luc1 infection, 2439 genes induced by Ad5/3luc1 infection, and 1251 genes induced by Ad5lucRGD infection, compared to unifected cells. Among these changes in gene expression, 783 changes were common to Ad5/3luc1 and Ad5luc1 infections, 266 were common to Ad5lucRGD and Ad5luc1 infections, and 185 changes in gene expression were common to Ad5/3luc1 and Ad5lucRGD infections. Interestingly, 89 changes in gene expression were common to all the three groups, suggesting a commonly affected pathway. This analysis represents a unique application of microarray to study vector-related issues.Furthermore, these studies demonstrate the utility of microarray for characterizing the biologic sequelae of host-vector interaction.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

et al. 2004

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“…In addition to treating cells with RIa antisense ODN, we have stably transfected PC3M cells with the MTRIa antisense vector, which contains the RIa cDNA (the N-terminal 210 nucleotides) in the antisense (Cho et al, 2001). This system avoids the problems inherent in oligonucleotide treatment, namely the delivery and stability of the oligonucleotide.…”

Section: Overexpression Of Ria Antisense Gene In Pc3m Cells Alters Thmentioning

confidence: 99%

Nuclear translocation of the catalytic subunit of protein kinase A induced by an antisense oligonucleotide directed against the RIα regulatory subunit

Neary

Cho‐Chung

2001

Oncogene

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The regulatory (R) subunits of cAMP-dependent protein kinase (PKA) are implicated in the regulation of cell proliferation and di erentiation. There are two isoforms of PKA that are distinguished by two types of R subunit, RI and RII. Evidence suggests that RI is associated with proliferation and RII is associated with cell di erentiation. Previous work in this laboratory has demonstrated that depletion of the RIa subunit by treatment with an antisense oligonucleotide (ODN) induces di erentiation in leukemia cells and growth arrest and apoptosis in epithelial cancer cells. Using the prostate cancer cell line PC3M as a model system, we have developed a cell line that overexpresses a retroviral vector construct containing the RIa antisense gene. This cell line has been characterized and the e ectiveness of the construct determined. In the work presented here, we demonstrate by immunocytochemistry that treatment with RIa antisense ODN induces translocation of the Ca subunit of PKA to the nucleus of PC3M prostate cancer cells. The translocation of Ca triggered by exogenous antisense ODN treatment mirrors that observed in cells endogenously overexpressing the antisense gene. Triggering the nuclear translocation of the Ca subunit of PKA in the cell may be an important mechanism of action of RIa antisense that regulates cell growth independent of adenylate cyclase and cellular cAMP levels. The nuclear localization of the Ca subunit of PKA may be an essential step in revealing the mechanism whereby this critical kinase regulates cell growth. Oncogene (2001) 20, 8019± 8024.

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“…In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. Indeed, antisense oligonucleotides have been shown to bind to many different proteins (15-17) and cause significant nonspecific effects (18)(19)(20). In addition, several RNA aptamers are known to bind to endogenous proteins and alter their function (21).…”

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confidence: 99%

Specificity of short interfering RNA determined through gene expression signatures

Semizarov

Frost

Sarthy

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

452

305

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Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.DNA microarray ͉ RNA interference ͉ gene knockdown T he use of RNA interference for inhibiting gene expression represents a powerful tool for exploring gene function (1-7), identifying and validating new drug targets, and treating disease (8, 9). The process of RNA interference is mediated by doublestranded RNA, which is cleaved by the enzyme DICER into 21-to 23-nt duplexes containing a 2-nt overhang at the 3Ј end of each strand. These duplexes are incorporated into a protein complex called the RNA-induced silencing complex (RISC). Directed by the antisense strand of the duplex, RISC recognizes and cleaves the target mRNA (for recent reviews, see refs. 1 and 10-13). Although long double-stranded RNAs (Ͼ30 nt) invoke an interferon response, short interfering RNAs (siRNAs) that resemble the products produced by DICER have been reported to specifically inhibit gene expression in many different mammalian cell lines (1-7). It has been shown that even single nucleotide mismatches between the antisense strand of the siRNA and target mRNA can abolish RNA interference (14). In addition, mapping of mRNA cleavage sites has revealed no cleavage sites outside of the region of complementarity (10). However, the specificity of siRNA at the cellular level remains to be comprehensively studied.For siRNAs to be a useful tool in gene knockdown experiments, it is critical that siRNA-mediated transcriptional silencing be specific. It is not enough to simply show that a control siRNA with a scrambled nucleotide sequence fails to knock down the protein of interest or produce the same cellular phenotype. Ideally, the siRNA must not cause any effects other than those related to the knock down of the target gene. There are several types of nonspecific effects that siRNA could potentially display. In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. I...

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Antisense DNAs as multisite genomic modulators identified by DNA microarray

Cited by 74 publications

References 29 publications

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

Nuclear translocation of the catalytic subunit of protein kinase A induced by an antisense oligonucleotide directed against the RIα regulatory subunit

Specificity of short interfering RNA determined through gene expression signatures

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