GENE ACTIVATION STUDIED BY IMMUNOLOGICAL METHODS

Gene activation can be studied at several levels: transcription (mRNA) , translation (proteins), or phenotypical alterations (functional acti vity or morphology). These levels can be studied in situ or biochemica lly by the use of specific probes for normal or altered DNA, mRNA, or proteins. immunological probes are potent tools for studies of alterat ions induced by xenobiotics in target organs. When the effects of xeno biotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ a nd biochemical techniques are necessary. Antibodies to normal or alter ed cellular constituents are used for identification, quantitation, an d cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-a cetylaminofluorene (AAF) forms DNA adducts, which can be identified im munologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentra tions in proliferating cells and preneoplastic foci in the livers of A AF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to ph enotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for st udies of gene activation. In mouse skin, the keratins K1 and K10 are o nly expressed in suprabasal, differentiating cells. BrdU pulse chase e xperiments combined with double immunofluorescence have revealed that K1 and K10 are sequentially turned on 18 to 24 hr after DNA synthesis and are followed by suprabasal migration. After a single application o f the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis. Keratin K1 and K10 were still turned on 18 to 24 hr after mitosis. The experiments demonstrated tha t TPA induced accelerated cell migration. Initiation of cell maturatio n after replication was not affected by TPA, while transition through the maturation program was significantly accelerated. Gene activation can also be studied with immunological probes to the proteins that reg ulate gene expression, such as growth factor receptors, signal mediato rs, and transcription factors. It must be kept in mind that immunologi cal detection reveals the presence of proteins, not their activity. Fu nctional activity may be related to intracellular localization: The lo calization of protein kinase C, c-fos, and c-mye proteins, for example , may be altered by their activation. In other cases, activation may b e detected by demonstration of phosphorylation, dimerization, or DNA b inding. Western blotting and immunofluorescence have demonstrated incr eased expression of the regulatory units of cAMP-dependent protein kin ase in rat liver after partial hepatectomy (PH) and when PH is combine d with dietary AAF for growth inhibition. immunological techniques may be powerful tools for characterization of gene activation. The specif icity of the reagents needs to be extensively controlled under the emp loyed conditions. For studies in whole tissue, immunohistochemical tec hniques give the additional opportunity to identify gene activation or expression in subpopulations and individual cells.