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Cloning and analysis of a FoxO transcription factor from Xiphophorus
Michael D Rudd1, Dennis A Johnston, Steven Kazianis
1Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park - Research Division, Smithville, TX 78957, USA.
Abstract:
Melanoma development in the fish Xiphophorus is determined, at least in part, by overexpression and activation of the Xmrk-2 oncogene, which triggers a variety of signal transduction pathways resulting in altered cell cycle control. We have begun analysing transcription factors which may link Xmrk-2 with regulation of cell proliferation or apoptosis. Towards this end, we have cloned an FKHR (FoxO sub-family) homolog from Xiphophorus maculatus. The isolated clone is a 2.7 kb cDNA encoding a predicted protein of 664 amino acids. The gene, which we have named FoxO5, maps to Xiphophorus Linkage Group XV. The protein product can be categorized within a branch of the FOXO sub-class, which includes: Danio rerio zFKHR (foxo5), Homo sapiens FKHR-L1 (FoxO3a) and Mus musculus FKHR2 (Foxo3). Notably, the Forkhead DNA binding domain, three Akt consensus phosphorylation sites and a carboxy-terminal minimal activation domain are each highly conserved. A mutated FoxO5 protein with disrupted Akt phosphorylation sites inhibits proliferation, but the wild-type protein fails to do so, when exogenously expressed in Xiphophorus cells derived from a melanoma. The same mutated protein predominantly localizes to the nucleus, yet the wild-type protein seldom does. Further characterization of Xiphophorus FoxO5 will contribute to understanding the molecular basis of carcinogenesis in these species.
Insights
Researchers identified a new gene, FoxO5, in Xiphophorus fish that may link the Xmrk-2 oncogene to cell proliferation and cancer. Understanding FoxO5
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Melanoma development in Xiphophorus fish is linked to Xmrk-2 oncogene activation.
- Signal transduction pathways and cell cycle control are affected by Xmrk-2.
- Transcription factors are being investigated for their role in linking Xmrk-2 to proliferation and apoptosis.
Purpose of the Study:
- To clone and characterize a Forkhead box O (FoxO) homolog in Xiphophorus maculatus.
- To investigate the role of this FoxO homolog in regulating cell proliferation and apoptosis in relation to the Xmrk-2 oncogene.
Main Methods:
- Cloning of a 2.7 kb cDNA encoding a predicted 664-amino acid protein, named FoxO5.
- Mapping of the FoxO5 gene to Xiphophorus Linkage Group XV.
- Comparative analysis of conserved domains (Forkhead DNA binding, Akt phosphorylation sites) with other species' FoxO proteins.
Main Results:
- Exogenous expression of a mutated FoxO5 (with disrupted Akt phosphorylation sites) inhibited proliferation in Xiphophorus melanoma cells.
- Wild-type FoxO5 failed to inhibit proliferation under the same conditions.
- Mutated FoxO5 predominantly localized to the nucleus, while wild-type FoxO5 seldom did.
Conclusions:
- Xiphophorus FoxO5 plays a role in regulating cell proliferation, potentially through nuclear localization and phosphorylation.
- Further characterization of Xiphophorus FoxO5 is crucial for understanding the molecular mechanisms of carcinogenesis in these fish.
- This study identifies a novel gene involved in the oncogenic pathway of Xiphophorus melanoma.