Date of Award

2003

Degree Type

Dissertation

Degree Name

Doctor of Pharmacy (PharmD)

First Advisor

Bingfang Yan

Abstract

The basic helix-loop-helix (bHLH) proteins are intimately associated with developmental events such as cell differentiation and lineage commitment. The HLH domain in the bHLH motif is responsible for dimerization whereas the basic region mediates sequence specific DNA binding. Human DEC, mouse STRA and rat SHARP proteins represent a new c lass of bHLH proteins. In each species, two members are identified with a sequence identity of>90% in the bHLH region and - 40% in the total protein, respectively. Based on sequence alignment and domain analysis, DEC/STRA/SHARP proteins show high s imilarities (- 40%) to Drosoph ila Hairy and E(spl) as well as the mammalian homologues (e.g., HES) in the bHLH domain. However, they lack the C-terminal WRPW motif which is present at all other Ha iry/E(spl)/Hes proteins and mediates transcription repression. These structural differences distinguish DEC/STRA/SHARPs from other bHLH proteins and indicate that they have dist inct biological functions. The purpose of this dissertation is to elucidate the oncogenic roles of DEC I and determine the molecular actions of DEC I on transcription regulation. Expression measurments demonstrate that DEC I is abundantly expressed in cancer tissues but not in adjacent nonmal tissues. In stable transfectants, DEC I inhibits cell proliferation, antagonizes serum deprivation-induced apoptosis, and selectively decreases the activities of several major caspases. Western blotting analyses identify that antiapoptotic protein surviving is a fu nctional mediator responsible for DEC I-directed antiapoptotic activity. DEC I and surviving exhibit a paralleled expression pattern in paired tumor-normal tissues. In co-transfection experiments, DEC I stimulates the survivin promoter, and this mechanism relies on the physical interactions with Sp I sites in the proximal promoter. In contrast, DEC I and its structurally related protein DEC2 show an inverted expression pattern in paired tumor-normal tissues. Forced expression of DEC I causes proportional decreases in the expression of DEC2 in stable transfectants. Co-transfection with DEC I represses the activity of a DEC2 promoter reporter by as much as 90%. The DEC I mediated transcription repression is achieved by direct binding to the E-box element in the proximal promoter of DEC2. The established activation of the survivin promoter provides a molecular explanation for its oncogenic involvement, and the differential activities toward DEC2 and survivin promoters establish that DEC I can act as a repressor or an activator depending on the genomic context of a target gene.

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