MOLECULAR CHARACTERIZATION OF ACF (ABERRANT CRYPT FOCI)

异常隐窝灶（ACF）是结肠癌的前兆。人和小鼠的ACR要么是增生性的，要么是增生性的，要么是发育不良的，这是一种基于形态学和它们形成肿瘤的潜力的分类。了解控制ACFs形成及其转化为完全恶性结肠病变的机制是本建议的重点。众所周知，近交系小鼠的遗传特性导致致癌物治疗后对结肠肿瘤形成的易感性或耐药性。我们将测试两个假设，试图定义决定差异易感性的遗传机制。特异性目的1：在不同结肠肿瘤易感性小鼠中形成的ACFs的恶性潜能是否源于靶细胞获得的特定基因突变补体？使用结肠癌致癌物偶氮甲烷，我们将在小鼠体内产生ACF群体。我们已经证明在耐药菌株中产生增生性ACF，但在癌症中没有进展。我们将对ACFs亚群进行详细的形态计量学和遗传分析，这将使我们能够理解为什么偶氮甲烷诱导的病变不能在耐药AKR小鼠中进展为肿瘤。激光捕获显微解剖将用于从单个隐窝中分离DNA，用于肿瘤相关基因的突变分析。1.1甲氧苄甲烷诱导的肿瘤易感和耐药小鼠ACF形成的时间过程是什么？1.2区分增生和发育不良ACF的功能特征是什么？1.3增生ACFs与发育不良ACFs在K-ras、A[C]、β -catenin、p53基因突变频率上是否存在差异？特异性目的2:ACFs的恶性潜能是否由分泌型磷脂酶A2的表达和信号传导控制，由Moml基因座编码？在影响肿瘤发生的遗传因素中，影响多发性肠肿瘤（Min）模型中肿瘤发生的因素是Moml的基因产物，一种钙依赖性非胰腺分泌磷脂酶（Pla2g2a [Pla2]）。sPla2在炎症中发挥作用，水解甘油脂的sn-2位置，释放花生四烯酸用于前列腺素合成。我们发现sPla2在小鼠克隆中的差异表达模式与肿瘤对偶氮甲烷的易感性呈负相关：a /J < SWR < AKR。这表明结肠sPla2也在化学诱导的肿瘤发生中发挥作用，与其在Min模型中的作用相当。我们打算探索sPla2影响敏感和耐药小鼠肿瘤形成的机制。2.1哪些结肠细胞类型产生sPla2/ sPla2的水平或功能在癌变诱导的ACF和肿瘤内部和附近是否有变化？2.2使用sPla2特异性抑制剂能逆转AKR对致癌物质的耐药性吗？2.3 sPla2在转基因A/J小鼠中过表达是否对偶氮氧甲烷诱导的肿瘤发生具有保护作用？

Aberrant crypt foci (ACF) is a precursor for colon cancer. Human and mouse ACR are either hyperplastic are either hyperplastic or dysplastic, a classification that is based on morphology and their potential to form tumors. Understanding mechanisms that govern formulation of ACFs and their conversion to fully malignant colonic lesions is the focus of this proposal. It is known that heritable characteristics of inbred mice lead to either susceptibility or resistance to formulation of colon tumors after carcinogen treatment. We will test two hypotheses that attempt to define genetic mechanisms that determine differential susceptibility. Specific aim 1: Does malignant potential of ACFs, formed in mice of differing colon tumor susceptibilities, result from the specific complement of gene mutations that target cells acquire? Using the colon carcinogen, azoxymethane, we will generate populations of ACF in mice. We have shown that hyperplastic ACF are produced in resistant strains, but fail to progress in carcinomas. We will conduct a detailed morphometric and genetic analysis of sub-populations of ACFs that will enable us to understand why azoxymethane-induced lesions fail to progress to tumors in resistant AKR mice. Laser capture micro-dissection will be used to isolated DNA from single crypts for mutational analyses of tumor related genes. 1.1 What is the time course of zoxymethane- induced ACF formation in tumor susceptible and resistant mice? 1.2 What functional characteristics distinguish hyperplastic and dysplastic ACF? 1.3 Are there differences between hyperplastic and dysplastic ACFs in the frequency of mutations in K-ras, A[C, beta-catenin and p53? Specific aim 2: Is malignant potential of ACFs controlled by expression of, and signalling by, the secretory phospholipase A2, encoded by the Moml locus? Among genetic factors that affect tumorigenesis factors that affect tumorigenesis in the multiple intestinal neoplasia (Min) model is the gene product of Moml, a calcium-dependent non-pancreatic secretory phospholipase (Pla2g2a [Pla2]). sPla2 plays a role in inflammation and hydrolyzes the sn-2 position of glycerolipids, releasing arachidonic acid for prostaglandin synthesis. We present evidence that sPla2 is differentially expressed in mouse clone in a patter inversely correlated with tumor susceptibility to azoxymethane: A/J < SWR < AKR. This suggests that colonic sPla2 also plays a role in chemically-induced tumorigenesis equivalent to its role in the Min model. We propose to explore the mechanism by which sPla2 affects tumor formation in sensitive and resistant mice. 2.1 What colon cell types produce sPla2/ Are there changes in sPla2 levels or function within and adjacent to carcinogen-induced ACF and tumors? 2.2 Can AKR resistances to carcinogen by reversed with the use of specific inhibitors of sPla2? 2.3 Can over- expression of sPla2 in transgenic A/J mice protect against azoxymethane- induced tumorigenesis?