Identification and Validation of a Gene Involved in Anchorage-Independent Cell Growth Control Using a Library of Randomized Hairpin Ribozymes

Welch, Peter J.; Marcusson, Eric G.; Li, Qi-Xiang; Beger, Carmela; Krüger, Martin; Zhou, Chen; Leavitt, Mark C.; Wong‐Staal, Flossie; Barber, James David

doi:10.1006/geno.2000.6230

Cited by 55 publications

(27 citation statements)

References 8 publications

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“…These retransformed cells regained transformation phenotypes such as anchorage-independent growth and tumor formation (17). 7 Interestingly, NR4A1 and NR4A2 were found again to be up-regulated in these retransformed cells and in the xenograft tumors derived from the retransformed cells, as compared with parental nontransformed HeLaHF.…”

Section: Discussionmentioning

confidence: 94%

“…Anchorage-independent growth is a hallmark of cell transformation, and soft agar colony formation assay is the most commonly used in vitro assay to assess anchorage-independent growth. Soft agar growth is also the distinguishing phenotype between parental HeLa and revertant HF cells in vitro (15,17,19). To examine the potential role of the NR4A2 in the differential phenotypes of HeLa and HF cells, two siRNAs against the gene were used to reduce the gene expression in HeLa cells, and the soft agar growth was then assessed.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

Ning¹,

Claassen²,

Yu³

et al. 2004

Cancer Research

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116

121

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HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/ NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, downregulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

Ning¹,

Claassen²,

Yu³

et al. 2004

Cancer Research

Self Cite

116

121

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“…For example, randomization of the 12-nucleotide DNA sequence encoding the two binding arms of the hairpin ribozyme generates a library of 4 12 or ϳ1.7 ϫ 10 7 distinct RNA ribozyme genes (16). Following delivery of the library into target cells via retroviral transduction and selection for a desired phenotype, ribozymes that target cellular genes involved in several important viral and oncogenic pathways have been identified (4,16,19,33). We report here the identification of hairpin ribozymes that suppress human immunodeficiency virus (HIV) in cell culture by using an in vivo selection scheme based on the Rz library and an HIV indicator cell line, CEM-GFP (10).…”

mentioning

confidence: 99%

Identification of Cellular Cofactors for Human Immunodeficiency Virus Replication via a Ribozyme-Based Genomics Approach

Waninger

Kuhen

et al. 2004

J Virol

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Ribozymes are small, catalytic RNA molecules that can be engineered to down-regulate gene expression by cleaving specific mRNA. Here we report the selection of hairpin ribozymes that inhibit human immunodeficiency virus (HIV) replication from a combinatorial ribozyme library. We identified a total of 17 effective ribozymes, each capable of inhibiting HIV infection of human CD4 ؉ cells. These ribozymes target diverse steps of the viral replication cycle, ranging from entry to transcription. One ribozyme suppressed HIV integration and transcription by inhibiting the expression of the Ku80 subunit of the DNA-activated protein kinase. Another ribozyme specifically inhibited long terminal repeat transactivation, while two additional ones blocked a step that can be bypassed by vesicular stomatitis virus G-protein pseudotyping. The function of Ku80 in HIV replication and its mechanism of action were further confirmed using short interfering RNA. Identification of the gene targets of these and other selected ribozymes may reveal novel therapeutic targets for combating HIV infection.

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“…These include (i) genes regulating the BRCA1 promoter (3), (ii) cellular genes mediating hepatitis C virus (HCV) internal ribosome entry site (IRES) activity (14), (iii) genes involved in anchorage-independent cell growth control (23), and (iv) genes involved in suppression of fibroblast transformation (16). Ribozymes that repeatedly conferred distinct cellular phenotypes were selected in these systems.…”

mentioning

confidence: 99%

Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation

Krüger

Beger

Welch³

et al. 2001

Molecular and Cellular Biology

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Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme ( Ribozymes are small catalytic RNA molecules that hybridize to complementary sequences of a particular target mRNA and that can be engineered to enzymatically cleave and destroy RNA transcripts in a sequence-specific fashion, thereby preventing expression and function of the corresponding gene product. Ribozymes have been studied in connection with a variety of diseases and human disorders as potential therapeutic molecules (for recent reviews see references 2, 9, 15, 19, and 22). Hairpin ribozymes fold into a two-dimensional hairpin structure, consisting of a small catalytic region with four helical domains (see Fig. 1A). Based on Watson-Crick base pairing, the ribozyme binding arms (helices 1 and 2) hybridize to sequences flanking the cleavage site (GUC) within the target RNA, thereby determining specificity of the recognized target sequence.Recently, we introduced a novel "inverse genomics" procedure based on a retrovirus hairpin ribozyme library with randomized target recognition sequences for gene discovery in different experimental systems. These include (i) genes regulating the BRCA1 promoter (3), (ii) cellular genes mediating hepatitis C virus (HCV) internal ribosome entry site (IRES) activity (14), (iii) genes involved in anchorage-independent cell growth control (23), and (iv) genes involved in suppression of fibroblast transformation (16). Ribozymes that repeatedly conferred distinct cellular phenotypes were selected in these systems. Single ribozyme candidates were identified, and the binding sequence flanking the GUC site required for ribozyme cleavage was exploited to identify partial sequence information of the target gene responsible for the observed phenotype. For the HCV IRES project, a cellular selection scheme was developed using a reporter system based on herpes simplex virus (HSV) thymidine kinase (TK) as a negative selectable marker under translational control of the HCV IRES (14). This cellular selection system allowed the identification of ribozymes that actively inhibited HCV IRES-mediated translation of HSV TK and thereby conferred a ganciclovir (GCV)-resistance phenotype. By using the ribozyme binding sequences, potential cellular cofactors for HCV IRES were discovered (14). For these experiments, Rz3ЈX, a hairpin ribozyme engineered against the minus-strand HCV replication intermediate at position 40 within the terminal 98-nucleotide (nt) (3ЈX tail)

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Identification and Validation of a Gene Involved in Anchorage-Independent Cell Growth Control Using a Library of Randomized Hairpin Ribozymes

Cited by 55 publications

References 8 publications

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

Identification of Cellular Cofactors for Human Immunodeficiency Virus Replication via a Ribozyme-Based Genomics Approach

Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation

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