MOLECULAR CHARACTERIZATION OF ACF (ABERRANT CRYPT FOCI)

异常隐窝病灶(ACF)是结肠癌的先兆。人类和小鼠的ACR要么是增生性的，要么是增生性的或异常的，这是一种基于形态和形成肿瘤的潜力的分类。了解ACFS的形成及其转化为完全恶性结肠病变的机制是本提案的重点。已知近交系小鼠的遗传特性导致对致癌物治疗后的结肠癌的易感性或抵抗力。我们将测试两个假说，这两个假说试图定义决定差异易感性的遗传机制。具体目标1：在不同结肠癌易感性的小鼠中形成的ACF的恶性潜能是否源于靶细胞获得的特定基因突变的补体？利用结肠癌致癌物质偶氮甲烷，我们将在小鼠身上产生ACF种群。我们已经证明，在耐药菌株中可以产生增生性ACF，但在癌症中却无法进展。我们将对ACF的亚群进行详细的形态计量和遗传分析，这将使我们能够理解为什么AKR耐药小鼠的偶氮甲烷诱导的损伤未能进展为肿瘤。激光捕获显微切割将用于从单个隐窝中提取DNA，用于肿瘤相关基因的突变分析。1.2增生性和非增生性ACF有哪些功能特征？1.3增生性和非增生性ACF在K-ras、A[C、β-catenin和P53基因突变频率上有无差异？特定目标2：ACFS的恶性潜能是否由MOM1基因编码的分泌型磷脂酶A2的表达和信号控制？在多发性肠道肿瘤(Min)模型中，影响肿瘤发生的遗传因素中有一种是MOM1的基因产物，它是一种钙依赖的非胰腺分泌型磷脂酶(Pla2g2a[PLA2])。SPLA2在炎症中起作用，它能分解甘油脂的sn-2位，释放花生四烯酸合成前列腺素。我们提出的证据表明，sPLA2在小鼠克隆中的差异表达与肿瘤对偶氮甲烷的敏感性呈负相关：A/J&lt；SWR&lt；AKR。这表明结肠sPLA2在化学诱导的肿瘤发生中的作用与其在Min模型中的作用相当。我们建议探索sPLA2影响敏感和耐药小鼠肿瘤形成的机制。2.1哪些类型的结肠细胞产生sPLA2/在致癌物诱导的ACF和肿瘤内及其周围是否有sPLA2水平或功能的变化？2.2使用sPLA2的特异性抑制剂能否逆转AKR对致癌物的抵抗？2.3在转基因A/J小鼠中过表达sPLA2能否保护AKR对抗偶氮甲烷诱导的肿瘤发生？

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?