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| Article |
| Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene |
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| R Nagaraja Rao1, Nancy B Stamm1, Keith Otto1, Steve Kovacevic2, Scott A Watkins1, Pam Rutherford1, Stephanie Lemke1, Kim Cocke1, Richard P Beckmann1, Keith Houck3, David Johnson1 and Barry J Skidmore1 |
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1Cancer Research Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285-0424, USA
2Research Technologies and Proteins, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285-0424, USA
3Sphinx Pharmaceuticals, A Division of Eli Lilly and Company, Durham, North Carolina, NC 27707, USA
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Correspondence to: R Nagaraja Rao, Lilly Corporate Center, Drop Code 0424, Indianapolis, IN 46285-0424, USA
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| Abstract |
| Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity. |
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| Keywords |
| cyclin D1; cdk4; transformation; cell models |
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| Abbreviations |
| cdk, cyclin-dependent kinase; D1.k4, cyclin D1 complexed with cdk4; D1-k4, cyclin D1 fused with cdk4; IP, immunoprecipitation; IB, immunoblot; REFs, rat embryo fibroblasts; Rb, retinoblastoma protein; FCS, fetal calf serum; tet, tetracycline |
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| Received 6 November 1998; revised 27 May 1999; accepted 3 June 1999 |
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| 4 November 1999, Volume 18, Number 46, Pages 6343-6356 |
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