The frequent loss of 8p sequences observed in human prostate tumors suggests that tumor suppressor genes (TSGs) critical for prostate tumorigenesis map to this chromosomal region. Our laboratory has determined that one or more TSGs likely map to proximal 8p, between markers D8S278-D8S255, within a region characterized by homozygous deletion and chromosomal breakage in human prostate tumors Moreover, preliminary work utilizing cDNA microarray, RT-PCR and Northern blot analysis has demonstrated that transcripts mapping to this region are absent or downregulated in primary tumor-derived human prostate cell lines deleted for one copy of proximal 8p. Based upon this work, we now hypothesize that candidate TSGs mapping to this region may be identified through the downregulation or absence of their corresponding transcripts in cells and tissues deleted for one copy of proximal 8p. We propose to employ an expression-based approach utilizing cDNA arrays and Northern blot analysis to test this hypothesis through the completion of three specific aims:
SPECIFIC AIM 1 : Produce a cDNA array for the proximal 8p-deleted region comprising all transcribed sequences currently binned between markers D8S278-D8S255;
SPECIFIC AIM 2 : Identify and verify differentially expressed genes by comparing the transcriptional expression profiles of the candidate TSGs between prostate-derived cell lines and xenografts that are either intact or deleted for one copy of proximal 8p sequences. Northern blot analysis will be used to verify the transcriptional downregulation of candidate tumor suppressor genes, and full-length cDNA clones corresponding to these transcripts will be isolated from a normal human prostate cDNA library;
SPECIFIC AIM 3 : Elucidate the contribution of specific genes to expression of the malignant phenotype through complementation studies to examine whether candidate genes identified in Aim 2 exhibit activities consistent with those of tumor suppressor genes. This work should identify TSGs critical for prostate tumorigenesis mapping to proximal 8p and provide a basis for understanding their biological functions and their role(s) in malignant transformation and progression.
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