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Transforming activity and mitosis-related expression of the AKT2 oncogene: evidence suggesting a link between cell
J Q Cheng1, D A Altomare, M A Klein
1Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Abstract:
The AKT2 oncogene encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling proteins. Recently, it was shown that AKT2 kinase activity can be induced by platelet-derived growth factor through phosphatidylinositol-3-OH kinase, suggesting that AKT2 may be an important signal mediator that contributes to the control of cell proliferation. We previously reported amplification and overexpression of AKT2 in human cancers. To investigate the transforming activity of AKT2, we used a retrovirus-based construct to express AKT2 in NIH3T3 cells. Overexpression of AKT2 was found to transform NIH3T3 cells, as determined by growth in soft agar and tumor formation in nude mice. The oncogenic activity of AKT2 was diminished by truncation of a 70-amino acid proline-rich region at the carboxyl-terminus. To facilitate the characterization of AKT2, we generated monoclonal and polyclonal antibodies against this protein. AKT2 was localized to the cytoplasm by cell fractionation experiments, immunocytochemistry, and immunofluorescence. Protein levels were more abundant in mitotic cells than in interphase cells. Western blot analysis of synchronized pancreatic cancer cells demonstrated that the expression level of AKT2 protein in mitotic cells is three to fivefold higher than in their interphase counterparts. A time-course study of phytohemagglutinin-stimulated lymphocytes revealed that AKT2 mRNA and AKT2 protein levels are highest 48-72 h after addition of mitogen, when cells are actively dividing. These data suggest that AKT2 could play a significant role in cell cycle progression and that the oncogenic activity of overexpressed AKT2 may be mediated by aberrant regulation of cellular proliferation.
Insights
The AKT2 oncogene promotes cell proliferation and cancer development. Its overexpression transforms cells, indicating a significant role in cell cycle progression and aberrant proliferation regulation.
Area of Science:
- Molecular Biology
- Oncology
- Cell Signaling
Background:
- The AKT2 oncogene encodes a protein-serine/threonine kinase involved in cell signaling.
- AKT2 kinase activity is induced by platelet-derived growth factor via phosphatidylinositol-3-OH kinase.
- AKT2 amplification and overexpression have been previously reported in human cancers.
Purpose of the Study:
- To investigate the transforming activity of the AKT2 oncogene.
- To characterize the role of AKT2 in cell proliferation and cell cycle progression.
- To generate antibodies for AKT2 protein characterization.
Main Methods:
- Retrovirus-mediated expression of AKT2 in NIH3T3 cells.
- Soft agar assays and tumor formation in nude mice to assess transformation.
- Western blot analysis, immunocytochemistry, and immunofluorescence for protein localization and expression levels.
- Analysis of synchronized pancreatic cancer cells and phytohemagglutinin-stimulated lymphocytes.
Main Results:
- Overexpression of AKT2 transformed NIH3T3 cells, leading to soft agar growth and tumor formation.
- Truncation of a carboxyl-terminal proline-rich region diminished AKT2's oncogenic activity.
- AKT2 protein is localized to the cytoplasm and is more abundant in mitotic cells (3-5 fold higher than interphase).
- AKT2 mRNA and protein levels peak during active cell division in stimulated lymphocytes.
Conclusions:
- AKT2 plays a significant role in cell cycle progression.
- The oncogenic activity of overexpressed AKT2 is likely mediated by aberrant regulation of cellular proliferation.
- AKT2 is a potential therapeutic target in cancers characterized by its overexpression.