Genomic Instability and Evolution of Drug Resistance

描述（由申请人提供）： DNA扩增经常在癌细胞和耐药细胞中观察到。DNA扩增中涉及的一个重要机制是断裂-融合-桥（BFB）过程。我们之前已经证明染色体脆性位点断裂会触发BFB周期。在许多CHO多药耐药细胞中，mdr 1的扩增与染色体脆性位点1 q31的断裂有关。为了了解mdrl扩增初始事件的分子机制，我们克隆了1 q31脆性位点DNA。我们发现该脆性位点含有一个新的基因，命名为脆性位点相关基因（fsa），Fas编码一个大于14 kb的mRNA。全长人FSA cDNA已被克隆。序列分析表明，它的mRNA是双顺反子，包含两个进化上保守的非重叠开放阅读框架（orf 1和orf 2）。虽然FSA mRNA的基础水平似乎在许多细胞类型中表达，但免疫组织化学分析揭示了两种非编码蛋白在许多器官的有丝分裂后、分化良好的上皮隔室中的共表达模式;包括结肠、乳腺、卵巢、前列腺和膀胱。这些发现表明FSA在调节哺乳动物上皮细胞的生长和分化中起重要作用。此外，在正常成熟上皮中，FSA蛋白似乎定位于细胞核中，而在上皮起源的恶性细胞中，FSA似乎主要是细胞质中。这些结果表明，上皮细胞转化过程中的核质洗牌。我们推测FSA表达升高可能诱导上皮细胞过早衰老并影响其对化疗药物的敏感性。我们提出了三个具体的目标，以进一步阐明该基因的结构/功能。在目的I中，我们建议严格重新调查FSA-orf 1和FSA-orf 2的细胞内定位，以及它们在许多组织来源的正常和恶性上皮细胞中的表达。在目的II中，我们建议通过转染来研究FSA-orf 1和FSA-ort 2在培养细胞中的功能，并验证上述假设。在目的III中，我们提出使用敲除策略来研究FSA-orf 1和FSA-orf 2的功能。我们期望从这些研究中获得重要的见解FSA在正常和恶性上皮细胞的细胞生长和分化的调节功能。

DESCRIPTION (provided by applicant): 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.