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扩增的一个重要机制是断裂-融合桥(BFB)过程。我们以前证明了染色体脆性部位的断裂触发了BFB循环。在许多CHO多药耐药细胞中，mdr1的扩增与染色体脆性部位1q31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1q31脆性位点DNA。值得注意的是，我们发现这个脆性部位含有一个新的基因，命名为脆性部位相关(FSA)基因，Fas编码一个大于14kb的mRNA。全长人FSA基因已被克隆。序列分析表明，该基因具有双顺反子结构，含有两个进化上保守的非重叠开放阅读框(orf1和orf2)。虽然基础水平的FSA mRNA似乎在许多细胞类型中都有表达，但免疫组织化学分析显示，这两种非编码蛋白在许多器官的有丝分裂后、分化良好的上皮室中共同表达；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发挥着重要作用。此外，在正常成熟的上皮细胞中，FSA蛋白似乎定位于细胞核，而在上皮起源的恶性细胞中，FSA似乎主要分布在细胞质中。这些结果表明，在上皮转化过程中，细胞核-细胞质发生了洗牌。我们推测，FSA表达升高可能会导致细胞过早衰老，并影响上皮细胞对化疗的敏感性。我们提出了三个特定的目标来进一步阐明该基因的结构/功能。在目标I中，我们建议重新研究FSA-orf1和FSA-orf2在细胞内的定位，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AIM II中，我们建议通过转染法研究FSA-orf1和FSA-ort2在培养细胞中的功能，并验证上述假说。在目标III中，我们建议使用基因敲除策略来研究FSA-orf1和FSA-ORF2的功能。我们期待从这些研究中获得对FSA在调节正常和恶性上皮细胞的生长和分化中的作用的重要见解。

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.