Cloning and Characterizing Genes Involved in Monoterpene Induced Mammary Tumor Regression

摘要

Monoterpene-induced/repressed genes were identified in regressing rat mammary carcinomas treated with dietary limonene using a newly developed method termed subtractive display. The subtractive display screen identified 42 monoterpene-induced genes comprising 9 known genes and 33 unidentified genes, as well as 58 monoterpene-repressed genes comprising 1 known gene and 57 unidentified genes. Several of the identified differentially expressed genes are involved in the TGFbeta signal tranduction pathway, as demonstrated by isolation of M6P/IGF2R and TGFbetaIIR. TGFbeta signaling and its downstream effects were investigated in perillyl alcohol-treated rat mammary carcinomas. RNA expression studies showed TGFbeta-related genes were induced and temporally regulated: first: c-jun and c-fos (transient), second: TGFbeta1: third: M6P/IGF2R, TGFbetaIIR, and TGFbetaIR; and fourth: smad3. In situ protein expression studies confirmed upregulation and demonstrated colocalization of these genes in epithelial cells. Smad2/Smad3 exhibited nuclear localization. A subpopulation of Smad2/Smad3 nuclei colocalized with a subpopulation of apopotic nuclei. RNA expression studies demonstrated the cell cycle and apoptosis related genes p21(cip1/WAF1), p27(Kip1), bax, bad, and annexin I were induced; cyclin E and cdk2 were repressed; and bcl-2 and P53 were unchanged. Characterization of cell growth and death parameters revealed apoptosis was induced prior to induction of cytostasis.