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Gene activation studied by immunological methods
H S Huitfeldt1, A Heyden, E Skarpen
1Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway.
Abstract:
Gene activation can be studied at several levels: transcription (mRNA), translation (proteins), or phenotypical alterations (functional activity or morphology). These levels can be studied in situ or biochemically by the use of specific probes for normal or altered DNA, mRNA, or proteins. Immunological probes are potent tools for studies of alterations induced by xenobiotics in target organs. When the effects of xenobiotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ and biochemical techniques are necessary. Antibodies to normal or altered cellular constituents are used for identification, quantitation, and cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-acetylaminofluorene (AAF) forms DNA adducts, which can be identified immunologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentrations in proliferating cells and preneoplastic foci in the livers of AAF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to phenotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for studies of gene activation. In mouse skin, the keratins K1 and K10 are only expressed in suprabasal, differentiating cells. BrdU pulse chase experiments 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 of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Investigating gene activation requires studying DNA, mRNA, and protein alterations using combined in situ and biochemical methods. These techniques help identify xenobiotic-induced changes in cellular processes and differentiation markers.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Gene activation studies can occur at transcriptional, translational, or phenotypical levels.
- Studying xenobiotic effects requires accounting for cellular heterogeneity within organs.
- Immunological probes are crucial for detecting xenobiotic-induced alterations in DNA, mRNA, and proteins.
Purpose of the Study:
- To explore methods for studying gene activation at multiple levels.
- To investigate the use of immunological probes for xenobiotic-induced alterations.
- To examine the impact of xenobiotics on DNA modifications and cellular differentiation.
Main Methods:
- Utilized in situ and biochemical techniques for studying gene activation.
- Employed immunological probes to identify and localize proteins and modified DNA.
- Combined 2-acetylaminofluorene (AAF) adduct identification with bromodeoxyuridine (BrdU) pulse labeling.
- Applied BrdU pulse chase experiments with double immunofluorescence to study keratin expression in mouse skin.
Main Results:
- Developed techniques to detect reduced DNA adduct concentrations in proliferating cells and preneoplastic foci in AAF-fed rats.
- Demonstrated that carcinogen-induced DNA modifications are linked to altered gene activation and neoplasia.
- Showed that xenobiotics can affect cellular differentiation, using keratin K1 and K10 as markers.
- Revealed sequential keratin expression and cell migration patterns following DNA synthesis and in response to tumor promoters like TPA.
Conclusions:
- Combined in situ and biochemical methods, particularly immunological probes, are essential for comprehensive gene activation studies.
- Xenobiotics can induce DNA modifications and alter cellular differentiation, impacting gene activation pathways.
- Understanding these molecular mechanisms is critical for assessing the toxicological and carcinogenic potential of xenobiotics.