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Identification of putative c-Myc-responsive genes: characterization of rcl, a novel growth-related gene
1Department of Medicine, Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
The c-Myc protein is a helix-loop-helix leucine zipper oncogenic transcription factor that participates in the regulation of cell proliferation, differentiation, and apoptosis. The biochemical function of c-Myc has been well described, yet the identities of downstream effectors are just beginning to emerge. We describe the identification of a set of c-Myc-responsive genes in the Rat1a fibroblast through the application of cDNA representational difference analysis (RDA) to cDNAs isolated from nonadherent Rat1a and Rat1a-myc cells. In this system, c-Myc overexpression is sufficient to induce the transformed phenotype of anchorage-independent growth. We identified 20 differentially expressed cDNAs, several of which represent novel cDNA sequences. We further characterized one of the novel cDNAs identified in this screen, termed rcl. rcl expression is (i) directly stimulated by c-Myc; (ii) stimulated in the in vivo growth system of regenerating rat liver, as is c-myc; and (iii) elevated in human lymphoid cells that overexpress c-myc. By using an anti-Rcl antibody, immunoblot analysis, and immunofluorescence microscopy, the Rcl protein was found to be a 23-kDa nuclear protein. Ectopic expression of the protein encoded by the rcl cDNA induces anchorage-independent growth in Rat1a fibroblasts, albeit to a diminished extent compared to ectopic c-Myc expression. These data suggest a role for rcl during cellular proliferation and c-Myc-mediated transformation.
Insights
Researchers identified a novel gene, rcl, that is regulated by the oncogenic transcription factor c-Myc. Overexpression of rcl promotes cell proliferation and anchorage-independent growth, suggesting its role in c-Myc-driven cancer.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- The oncogenic transcription factor c-Myc regulates cell proliferation, differentiation, and apoptosis.
- While c-Myc's function is known, its downstream effectors are not fully identified.
Purpose of the Study:
- To identify novel c-Myc-responsive genes using cDNA representational difference analysis (RDA).
- To characterize the function of a newly identified gene, rcl, in cellular proliferation and transformation.
Main Methods:
- cDNA representational difference analysis (RDA) was used to compare gene expression in Rat1a fibroblasts with and without c-Myc overexpression.
- Quantitative analysis of differentially expressed cDNAs, including novel sequences.
- Characterization of rcl expression, protein localization, and functional effects on cell growth.
Main Results:
- Identified 20 differentially expressed cDNAs, including novel sequences.
- Identified and characterized a novel cDNA, rcl, directly stimulated by c-Myc.
- Rcl is a 23-kDa nuclear protein whose expression is elevated in human lymphoid cells overexpressing c-myc.
- Ectopic expression of rcl induces anchorage-independent growth in Rat1a fibroblasts.
Conclusions:
- Rcl is a novel c-Myc-responsive gene involved in cellular proliferation.
- Rcl plays a role in c-Myc-mediated cellular transformation and anchorage-independent growth.