DISRUPTION OF CELL CYCLE GENES IN BREAST CANCER

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DESCRIPTION: The mRNA cap binding protein, eIF4E, plays an essential role in the control of cell proliferation by regulating events at the G1-S phase transition and is regulated at multiple levels. Abnormally high levels of eIF4E are required to maintain the phenotype of breast cancer cells. The levels of eIF4E in biopsies of breast cancer and breast cancer cell lines are increased relative to benign fibroadenomas and control cells. A direct role for eIF4E in breast cancer is evidenced by studies of mammary carcinoma cells (MDA-435) exhibiting a 50 percent decrease in eIF4E expression, due to an antisense construct, that have a markedly reduced ability to produce tumors in nude mice. The applicants will use dominant negative mutants of eIF4E to test the hypothesis that overexpression of eIF4E causes breast cancer. In addition, they will use structure based site directed mutagenesis and random mutagenesis with screening to identify eIF4E mutants that have a high affinity for mRNA to learn more about mRNA binding. Mutants may be used in gene therapy of cancer (dominant negatives), nucleic acid based vaccines, or in biotechnology. Specific objectives are: (A) To determine (1) the effect of expressing dominant negative forms of eIF4E in breast cancer cells and (2) the effect of overexpressing wild-type and high affinity mutants of eIF4E in nonmalignant breast epithelial cells. Effects on eIF4F complex formation, cell cycle perturbations associated with increased proliferation, colony growth in soft agar, and tumor formation in nude mice will be determined. (B) To determine what residues in specific loops adjacent to the mRNA binding site of eIF4E can alter the affinity for mRNA. (C) To use random mutagenesis, a bacterial surface display system and fluorescent m7G probes to identify mutations of eIF4E that bind mRNA caps with high affinities. (D) to initiate cocrystal growth studies, with m7GpppG and m7G capped oligoribonucleotides, of several mutants that bind mRNA with a higher affinity than wild-type eIF4E.