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 (Hep G2) and rats (MH1C1) is presented. In comparison to a similar procedure rep orted 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 mug/ml) of proteinase K. The exclusive localiza tion of transcripts in the cytoplasm of hepatoma cells with the absence of nuclear staining and the completely negative sense controls confirm the spe cificity of the method. The marked differences in signal intensity between the results of albumin and p-actin mRNAs which are of high abundance in con trast to moderate to low abundance of peroxisomal mRNAs show the high sensi tivity and the wide range of applicability of our protocol. This is also co nfirmed by divergent results of treatment of hepatoma cell lines with clofi brate 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 ana lysis of total RNA extracts using P-32-labeled probes. Thus the protocol pr esented here provides a sensitive tool for ISH localization of mRNAs encodi ng peroxisomal proteins. In combination with immunocytochemistry it may be useful to monitor intercellular differences in expression levels of specifi c mRNAs in correlation with the abundance of structurally divergent forms o f peroxisomes (tubular versus spherical) and their importance in the biogen esis of peroxisomes.