Although it is generally believed that carcinogens act in part by activating unexpressed genes, it has been difficult in practice to study this phenomenon. We have developed an approach which takes advantage of the fact that mouse S49 cell do not normally express either metallothionein I (MT-I) or metallothionein II (MT-II), although the genes are intact. Treatment with four different carcinogens can activate the expression of one or both of these genes. We plan to evaluate the role of cis and trans effects in carcinogen-activation of MT expression. We will use parental S49 cells (MT) and cell lines derived from carcinogen treatment which MT-I+/MT-II-, MT-I-/MT-II+, or MT-I+/MT/II. Many of our variants are unique since they represent the only known mammalian cells which express a single metallothionein, and some show constitutive MT expressions which is not stimulated by Cd++. We will use transient expression assays to study the role of trans factors in carcinogen-induced MT-I and MT-II expression following the introduction of plasmids bearing MT-I or MT-II fusion genes (chloramphenicol acetyl-transferase (CAT ) or luciferase). These studies will be extended by performing gel retardation assays, DNA footprinting, and in vitro transcription studies. We plan to purify and characterize any trans factors which are activated by carcinogens. We will use an in vitro amplification protocol and direct sequencing techniques to evaluate the role of cis regulatory mutations in expression: the 5' flanking regions of MT-I and MT- II from wild type S40 cells and variants with different patterns of MT expression will be sequenced to identify potential regulatory mutations Mutant MT regulatory regions will be cloned directly and analyzed in transient expression assays, gel retardation studies, footprinting studies, and in vitro transcription assays. If all S49 cells regardless of MT expression status support transient expression of MT fusion genes and MT+ lines do not show 5'regulatory mutations, we will evaluate the relative importance of other cis effects (e.g., chromatin changes) by comparing the induced variant frequency (MT- MT+) with the frequency of G A transitions (analogous to the change seen in H-ras activation). By take advantage of this unique collection of carcinogen-induced MT variants, this project will provide an exciting and highly important analysis of how carcinogens effect changes in gene expression. The combination of cells with different phenotypes and the ability to analyze cis effects directly in concert with changes in trans regulation provide an unusual opportunity to study molecular mechanisms to carcinogen action.
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| Aguilar-Cordova, E; Lebovitz, R M; Lieberman, M W (1991) Analysis of the role of 5' regulatory mutations in the activation of quiescent metallothionein genes after carcinogen treatment. Gene Expr 1:217-21 |
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