MOLECULAR CHARACTERIZATION OF ACF (ABERRANT CRYPT FOCI)

异常隐窝灶（ACF）是结肠癌的前兆。人类和小鼠 ACR 要么是增生性的，要么是增生性的或发育异常的，这是一种基于形态学及其形成肿瘤的潜力的分类。了解控制 ACF 的形成及其转化为完全恶性结肠病变的机制是该提案的重点。众所周知，近交小鼠的遗传特征导致在致癌剂治疗后对结肠肿瘤制剂的易感性或抗性。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。具体目标1：在不同结肠肿瘤易感性的小鼠中形成的ACF的恶性潜力是否是由靶细胞获得的基因突变的特定互补导致的？使用结肠致癌物氧化偶氮甲烷，我们将在小鼠体内产生 ACF 群体。我们已经证明，增生性 ACF 在耐药菌株中产生，但在癌症中却无法进展。我们将对 ACF 亚群进行详细的形态测量和遗传分析，这将使我们能够了解为什么氧化偶氮甲烷诱导的病变无法在耐药 AKR 小鼠中进展为肿瘤。激光捕获显微切割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 在肿瘤敏感和耐药小鼠中，Zoxymacet 诱导 ACF 形成的时间过程是怎样的？ 1.2 增生性和不典型增生性 ACF 的功能特征是什么？ 1.3 增生性和不典型增生性 ACF 中 K-ras、A[C、β-catenin 和 p53 的突变频率是否存在差异？具体目标 2：ACF 的恶性潜能是否受 Moml 位点编码的分泌型磷脂酶 A2 的表达和信号传导控制？在影响多发性肠肿瘤（Min）模型中肿瘤发生的遗传因素中，影响肿瘤发生的因素是Moml的基因产物，Moml是一种钙依赖性非胰腺分泌性磷脂酶（Pla2g2a [Pla2]）。 sPla2 在炎症中发挥作用，并水解甘油脂的 sn-2 位，释放花生四烯酸用于前列腺素合成。我们提供的证据表明，sPla2 在小鼠克隆中差异表达，其表达方式与肿瘤对氧化偶氮甲烷的敏感性呈负相关：A/J < SWR < AKR。这表明结肠 sPla2 在化学诱导的肿瘤发生中也发挥着与其在 Min 模型中的作用相同的作用。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​​些结肠细胞类型产生 sPla2/致癌物诱导的 ACF 和肿瘤内部和附近的 sPla2 水平或功能是否发生变化？ 2.2 使用 sPla2 特异性抑制剂可以逆转 AKR 对致癌物的耐药性吗？ 2.3 转基因 A/J 小鼠中 sPla2 的过度表达能否预防氧化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?