Inducible expression of dominant negative insulin-like growth factor I receptor in MCF-7 breast cancer cells |
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Authors: | Modha Geetanjalee Blanchard Anne Sidorchuk Janine Venditti Marcello Shiu Robert Myal Yvonne |
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Institution: | (1) Department of Physiology, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada;(2) Department of Pathology, Faculty of Medicine, University of Manitoba, D212-770 Bannatyne Avenue, R3E 0W3 Winnipeg, MB, Canada |
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Abstract: | The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development.
In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in
the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression
to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow
us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I
receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPhCMV-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene
expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably
transfected doxycycline-induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I
stimulated cell proliferation diminished (twofold) in doxycycline-induced cells compared to uninduced cells, demonstrating
that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly,
autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was
not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other
than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly
examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells. |
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Keywords: | IGF-IR dominant negative tetracycline-inducible system mammary epithelial cells |
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