DNA amplification is frequently observed in cancer cells and in drug-resistant cells. One important mechanism involved in DNA amplification is the breakage-fusion-bridge (BFB) process. We previously demonstrated that chromosomal fragile site breakage triggers the BFB cycle. In many CHO multidrug-resistant cells, amplification of mdr1 is associated with the breakage of chromosomal fragile site 1q31. To learn the molecular mechanism underlying the initial events of mdrl amplification, we have cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contains a novel gene, designated as fragile site associated (fsa) gene, Fas encodes a mRNA of greater than14 kb. Full-length human FSA cDNA has been cloned. Sequence analyses revealed that its mRNA is bicistronic and contains two evolutionarily conserved nonoverlapping open reading frames (orf1 and orf2). While basal levels of FSA mRNA seem to express in many cell types, immunohistochemical analyses revealed a co-expression pattern of the two off-encoded proteins in the post-mitotic, well-differentiated epithelial compartments of many organs; including colon, mammary glands, ovary, prostate and bladder. These findings suggest that FSA plays important roles in, regulating mammalian epithelial growth and differentiation. Moreover, in normal mature epithelia, the FSA protein seems to be localized in nuclei, whereas in the malignant cells of epithelial origins, FSA seems to be mainly cytoplasmic. These results suggest a nucleus-cytoplasm shuffling during epithelial transformation. We hypothesize that elevated FSA expression may induce premature senescence and affect drug sensitivities to chemotherapy of epithelial cells. We propose three specific aims to further elucidate the structure/function of this gene.
In Aim I, we propose to critically re-investigate the intracellular localizations of FSA-orf1 and FSA-orf2, and their expression in normal and malignant epithelial cells from many tissue sources.
In Aim II, we propose to investigate the function of FSA-orf1 and FSA-ort2 in cultured cells by transfection and to test the above-mentioned hypothesis. And in Aim III, we propose to investigate the function of FSA-orf1 and FSA-orf2 using the knock out strategies. We anticipate from these studies to gain important insights into the function of FSA in the regulation of cell growth and differentiation in normal and malignant epithelial cells.
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