Student: Johanna Carroll '03

Supervisor: Stephen M. Festin, Assistant Professor of Biology

Project title: Establishing a system to test the influence of MAPkinase on AFP’s ligand-independent activation of the estrogen receptor.

Project Summary: Alphafetoprotein (AFP) is a protein which has been shown to stimulate the estrogen receptor through ligand-independent activation when overexpressed in HepG2 cells. In an attempt to elucidate the pathway by which AFP carries out its activity on the cell, we worked to establish a system to test the effects of MAPkinase inhibitors on AFP’s activation of the estrogen receptor. In order to do this we needed to use an overexpression system which was not affected by the interruption of the MAPkinase pathway. We chose to use plasmids under the control of a pLEN promoter, one which has been shown to be unaffected by the MAPkinase patheway. We transfected HepG2 cancer cells with an ERE-TK-Luciferase plasmid, an ER-a plasmid under the control of a pLEN promoter and b-gal plasmid also under the control of the pLEN promoter. The cells were then treated with AFP and estradiol in an attempt to establish that the system exhibited both ligand-independent activation and ligand-dependent activation of the estrogen receptor. We found that transfected cells exhibited ligand-dependent activation of the estrogen receptor when treated with estradiol; however, the results of the ligand-independent stimulation of the estrogen receptor due to AFP treatment remain inconclusive. Our experiments suggest however, that the pLEN system could be used to effectively test the effects of MAPkinase inhibitors on AFP’s activation of the estrogen receptor. Stipend support for J.C. was provided by an award from Bristol-Myers Squibb.

Student: Matt Child '04

Supervisor: Stephen M. Festin, Assistant Professor of Biology

Project title: AFP Stimulation of Estrogen Receptor at the C3 Promoter.

Project Summary: Alphafetoprotein (AFP), a member of the human albumin family produced during fetal development, and peptides derived thereof are known to arrest the growth of breast cancer cells in vitro and in vivo. AFP peptides are known to modulate estrogen receptor action through a ligand-independent mechanism in HepG2 hepatoma cells expressing the estrogen receptor. The purpose of this study was to conduct comparative analysis of two estrogen responsive promoters, one simple and one complex. Using promoter sequences cloned into luciferase reporter in a plasmids, the activity of a simple 13 bp palindromic estrogen response element (ERE) and a complex multiple non-palindromic ERE containing complement C3 promoter were compared. Our results indicate that ligand-independent activation of estrogen receptor by AFP peptides is not limited to a simple ERE containing promoter as previously demonstrated, but is also detected at the complex C3 promoter. These results further support evidence that AFP peptide activity is mediated through the N-terminal activation function-1 (AF-1) of the estrogen receptor by a mechanism different from that of estrogen mimics such as Tamoxifen, a drug currently in used in the treatment of estrogen receptor positive breast cancer. References: (1) Jacobsen, H. I. et al., Cancer Research, 50, 415-420, 1990. (2) Richardson et al., Amer. J. of Epidemiology, 148, 719-727, 1998. (3) Guancial, E. 2000. (4) Métivier et al., Mol Endocrinol, 15(11):1953-1970, 2001. Stipend support for M.C. was provided by an award from Merck-AAAS.