Id2 re-expression results in the downregulation of p27KIP1 and tumorigenesis in dermal fibroblasts
Trabosh, Valerie Anne.
Thesis (Ph.D.)--Georgetown University, 2009.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins for the retinoblastoma (Rb) tumor suppressor family involved in proliferation, cell cycle progression and differentiation. Tumorigenesis being the natural manifestation of unregulated cell cycle progression, it was imperative to determine the role of Id2 in tumor formation and the mechanism by which it regulates cell cycle and proliferation. This mechanism has not been previously explored with an Id2-null background, lending novelty to our studies. Id2-null dermal fibroblasts were derived from knockout mice pups and cultured under standard NIH 3T3 conditions. Id2 was reintroduced into the system through retroviral transduction and monitored for phenotype. There was a drastic increase in proliferation upon Id2 re-expression and a corresponding increase in the percentage of cells in S-phase. Additionally, introduction of Id2 caused the dermal fibroblasts to develop transformed properties such as the ability to proliferate in low serum, form foci, and initiate tumor formation in vivo. Following a comprehensive screen of several cell cycle proteins, it was determined that Id2 was able to produce such effects because it can downregulate the p27KIP1 promoter and subsequent p27KIP1 expression. The Id2-mediated sequestration of E-proteins (E12/E47) from the p27KIP1 promoter at the fourth E-box position was responsible for the decreased expression that is seen in the dermal fibroblasts. Mutational analysis of Id2 demonstrated that the HLH domain was important for tumorigenesis and p27KIP1 downregulation. Our findings suggest a novel role for Id2 as an oncogene capable of mediating tumorigenesis through the downregulation of p27KIP1.
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