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  • Original Paper
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Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene

Oncogene volume 18, pages 6343–6356 (1999)Cite this article

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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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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Acknowledgements

We thank Bruce Glover, for oligonucleotides; Pam Rockey, for DNA sequencing; Julia Enkema, for assistance with the surgical implantation of the tetracycline pellets; Mike Esterman, for help with capture and analysis of video images of soft agarose colony formation assays and for preparation of custom NIH Image macros; Jeff Dixon (Sphinx Pharmaceuticals), for purified cyclin D1.cdk4 complex; and Robert Swift for Ha-rasG12V and c-myc plasmids; and Beverly Teicher, for reviewing the manuscript. Special thanks are due to Mei Lai for a thorough and a critical review of the manuscript. We thank John Sedivy for permission to cite his unpublished results.

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Authors and Affiliations

  1. Cancer Research Division, A Division of Eli Lilly and Company, Lilly Research Laboratories, 46285-0424, Indianapolis, IN, USA

    R Nagaraja Rao, Nancy B Stamm, Keith Otto, Scott A Watkins, Pam Rutherford, Stephanie Lemke, Kim Cocke, Richard P Beckmann, David Johnson & Barry J Skidmore

  2. A Division of Eli Lilly and Company, Research Technologies and Proteins, Lilly Research Laboratories, 46285-0424, Indianapolis, IN, USA

    Steve Kovacevic

  3. A Division of Eli Lilly and Company, Sphinx Pharmaceuticals, Durham, 27707, North Carolina, NC, USA

    Keith Houck

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Rao, R., Stamm, N., Otto, K. et al. Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene. Oncogene 18, 6343–6356 (1999). https://doi.org/10.1038/sj.onc.1203009

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