2006

Abstract:

Sp1 and Sp3 are founding members of the growing Sp/XKLF (specificity protein/ Krüppel-like factor) family that comprises more than 20 site-specific transcription factors. This family is united by three conserved Cys2His2 zinc finger motifs that form their DNA binding domain recognizing similar GC and GT boxes. Sp1 and Sp3 are ubiquitously expressed in mammalian cells, and are obligatory to the transcription of hundreds of genes to regulate cell growth, cell cycle progression, hormonal activation, and apoptosis. There has been increasing evidence that dysfunction of Sp1 and other Sp proteins correlates with malignant phenotype of cancer cells. Higher expression and elevated transcriptional activity of Sp1 have been found in pancreatic cancer, breast cancer, gastric carcinoma, and thyroid carcinoma.

A recent study by Justin McCormick and associates at the Carcinogenesis Lab, MSU, indicated that Sp1 and Sp3 were expressed at 8-18 fold higher levels in more than 60% of the fibrosarcoma cell lines than that found in normal human skin fibroblasts. More significantly, when the expression of Sp1 and Sp3 was successfully reduced to near normal levels by RNA ribozyme, their tumor forming ability in athymic mice was either substantially weakened or completely eliminated. Our joint systems biology approach with Justin McCormick and the Carcinogenesis Lab aims to understand and predict, in quantitative detail, (1) how the steady-state level of Sp1 and Sp3 and their gene regulatory function are modulated in normal human cells, and (2) how their cellular functions are altered in cancer cells. We first modeled the promoter activity of Sp1 and Sp3 in various genes through a system of differential equations, in which the promoter occupancy, the RNA polymerize II binding, and RNA synthesis initiation are largely determined by competition of Sp1 and Sp3 on the proximal cognate sites.

The combined transactivation potency of these factors is controlled by the ratio of Sp1 and Sp3 concentrations, their individual and cooperative functions, and the number and relative location of binding sites. Statistical mechanics provided a framework for the computation of the occupancy probability at equilibrium for each Sp protein in the thermodynamic model. This modeling effort was then extended to the study of the stability of Sp proteins at equilibrium by a large system of differential equations, in which Sp protein levels are assumed to be modulated by their auto-regulation and cross-regulation loop. The dynamical complexity caused by the time delay inherited from mRNA and protein syntheses, and transcription factor transport, will also be discussed in a system of functional differential equations. This will help us to further incorporate various signaling pathways that perturb Sp protein function through post-translational modification in our model. The condensed localization of Sp proteins in the nucleus of cancer cells induced by these secondary modifications will be studied in our future work using integral-differential equations and reaction-diffusion equations.

Ultimately, our joint effort is expected to quantitatively examine our hypothesis that the auto-regulation and cross-regulation of Sp1 and Sp3, and their nuclear organization, are the key mechanisms that control their steady-state levels in non-tumor-forming cells, and that, in the cancer cells under study, sustained activation of Ras-mediated pathways that target Sp1 phosphorylation, or the lack of wild type p53 to form p53-Sp1 complex, is responsible for the over-expression and elevated activity of Sp1 and Sp3 and various proteins critical for malignant transformation.