MOLECULAR CHARACTERIZATION OF ACF (ABERRANT CRYPT FOCI)

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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?