CARCINOGENESIS OF COOKED FOOD MUTAGENS

University Of Calif-Lawrnc Lvrmr Nat Lab, Livermore CA

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Abstract

DESCRIPTION (provided by applicant): We propose to test the hypothesis that heterocyclic amine carcinogens (HAs) induce mutations in genes within cells of specific target tissues that are critical to oncogenic progression. Our goals are to identify and characterize these genes using a rat mammary gland carcinogenesis model, and to relate these findings to tumor progression and to the HA which induced the tumors. We propose three specific aims. The goal of the First Aim is to locate regions of gain/loss in DNA from mammary tumors induced by PhIP, MeIQx, IQ, and MeIQ. We will accomplish this by extracting DNA from rat tumors known to be induced by these heterocyclic amine carcinogens, and locate chromosome regions gained, lost or amplified in the tumors using comparative genomic hybridization (CGH). The goal of the Second Aim is to determine which genes are expressed in the chromosomal regions identified in Aim 1 in normal mammary tissue, and then to determine which of those genes are lost, amplified or mutated in the same chromosomal regions in rat mammary tumors. To accomplish this, normal rat mammary cDNA libraries will be hybridized to normal rat metaphase cells. We will microdissect the chromosome regions of these metaphase cells that were shown to be involved in HA-induced tumorigenesis under Aim 1, then sequence the recovered genes and place them on microarrays. mRNA isolated from each tumor will then be hybridized to the microarrays to determine which genes appear over- or underexpressed. Genes that appear to be amplified or lost will be sequenced to identify potential mutations that change DNA consensus sequences as well as protein coding regions that play an important role in HA-induced tumorigenesis. The goal of the Third Aim is to analyze the progression of genetic changes in mammary tumors. This will be accomplished by microdissecting small groups of cells from paraffin-embedded tumor sections, perform CGH on DNA isolated from these cells, and compare these data with CGH results obtained from total tumor DNA. We will also characterize the presence or absence of specific gene deletions and mutations discovered in Aim 2 using PCR and microarray analysis on DNA obtained from tumor sections. By analyzing cells at increasing distances from the center of tumors we will be able to identify and characterize the cellular events that drive tumor progression.