The MCF10 xenograft model consists of a number of variants derived from a single patient which include normal immortalized breast epithelial cells, estrogen responsive premalignant variants, a variant which forms rapidly growing but preinvasive ductal carcinoma in situ, and estrogen independent malignant variants in immune deficient mice. This panel of human breast cell lines, on a common genetic background, representing multiple events in breast disease will be used for comparative studies to identify genetic alterations which occur with progression. We hypothesize that E2 independent malignant lines may constitutively express proteins which are altered by E2 in the premalignant stages (E2 accelerates progression of MCF10AT1 xenografts). Thus, first priority will be to identify proteins expressed constitutively in malignant variants which are also induced by E2 in premalignant MCF10AT1 cells. It is proposed to look at relative levels of proteins rather than nucleic acids. The methods to be used to monitor protein expression involve the use of nonporous reversed phase HPLC separations which can rapidly separate large numbers of proteins from whole cell lysates and provide efficient recovery of those proteins in the liquid phase for further analysis. The method will be combined with a first dimension separation using liquid phase isoelectric focusing (IEF) to prefractionate proteins according to pI. The result is a 2-D protein map analagous to 2-D gel electrophoresis where the expression of hundreds of proteins can be imaged and monitored. Matrix-assisted Laser Desorption/Ionization time-of- flight mass spectrometry will mass size each target protein and digestion by trypsin or Glu-C will generate a peptide map. The molecular weight and peptide maps can be used to identify each protein and electrospray ionization mass spectometry will be used to pinpoint modifications such as missense mutations and post- translational events such as phosphorylation. The MCF10 system provides a direct means to test the cause and effect relationship of the detected change to the malignant phenotype. We will use the novel technique of gene conversion to inhibit gene expression or introduce missense mutations in cells at defined stages of progression and to monitor the effect of these alterations on progression as determined by stage of disease formed in xenografts.
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