Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization. Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs.
A non-radioactive in situ hybridization (ISH) protocol for localization of mRNAs encoding peroxisomal proteins in hepatoma cell lines from humans (HepG2) and rats (MH1C1) is presented. In comparison to a similar procedure reported for tissue sections, the cell culture preparations require only brief fixation in 4% paraformaldehyde and their permeabilization is achieved by a very low concentration (1 microg/ml) of proteinase K. The exclusive localization of transcripts in the cytoplasm of hepatoma cells with the absence of nuclear staining and the completely negative sense controls confirm the specificity of the method. The marked differences in signal intensity between the results of albumin and beta-actin mRNAs which are of high abundance in contrast to moderate to low abundance of peroxisomal mRNAs show the high sensitivity and the wide range of applicability of our protocol. This is also confirmed by divergent results of treatment of hepatoma cell lines with clofibrate and cetaben on mRNA levels of catalase and acyl-CoA oxidase. The ISH results of drug treatment of cell lines are confirmed also by slot blot analysis of total RNA extracts using 32P-labeled probes. Thus the protocol presented here provides a sensitive tool for ISH localization of mRNAs encoding peroxisomal proteins. In combination with immunocytochemistry it may be useful to monitor intercellular differences in expression levels of specific mRNAs in correlation with the abundance of structurally divergent forms of peroxisomes (tubular versus spherical) and their importance in the biogenesis of peroxisomes.
Kovacs
W
W
Institute of Medical Chemistry, University of Vienna, Währingerstrasse 10, 1090 Vienna, Austria.
Stangl
H
H
Völkl
A
A
Schad
A
A
Dariush Fahimi
H
H
Baumgart
E
E
D010129:4-Aminobenzoic Acid / Q000494:pharmacology*; D018528:ATP-Binding Cassette Transporters; D042962:Acyl-CoA Oxidase; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002374:Catalase / Q000235:genetics*; D018929:Cell Culture Techniques; D002994:Clofibrate / Q000494:pharmacology*; D004076:Digoxigenin; D006801:Humans; D000960:Hypolipidemic Agents / Q000494:pharmacology*; D017403:In Situ Hybridization / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D020675:Peroxisomes / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured; D062366:para-Aminobenzoates
C063305:ABCD3 protein, human; D018528:ATP-Binding Cassette Transporters; C489272:Abcd3 protein, rat; D000960:Hypolipidemic Agents; D008565:Membrane Proteins; D012333:RNA, Messenger; D062366:para-Aminobenzoates; C018246:cetaben; D010088:Oxidoreductases; D002374:Catalase; D042962:Acyl-CoA Oxidase; D002994:Clofibrate; D004076:Digoxigenin; D010129:4-Aminobenzoic Acid