Uptake and effects of antisense oligodeoxynucleotides specific for rat metallothionein -1 and -2 mRNA in H4 rat hepatoma cells in culture.

Metallothionein is an ubiquitous metal-binding protein which is inducible by various stimuli such as Cd. Cu, Zn, dexamethasone or various "stress" factors. In rat liver, there m two isoforms of metallothionein (MT-I and MT-2) but i t is unknown if they have separate functions [ I ] . Antisense oligodeoxynucleotides (ODNs) are commonly used to reduce the synthesis of a protein in order to study its function. This approach relies on the specific hybridisation of a short synthetic DNA strand with the complementary mRNA sequence. Formation of this duplex can suppress the translation of the protein 121. The aim of the present work was to use specific ODNs to selectively inhibit the synthesis of each MT isoform in cells in culture with the future objective of studying their function. The ODNs 13.41 were systematically designed by computer according to the following criteria: -the sequences were complementary to the 5' UTR of the mRNAs including at least part of the AUG start codon; the length of the ODNs was between 15 and 30 bases; to ensure specificity of the probes, the percentage of homology between the ODNs complementary sequence for each MT isoform and the other isoform mRNA was less than 60%; -the percentage of cytosines plus guanines (C+G) and the melting temperature (Tm) of the ODNs were as high as possible. The final sequences were further analysed for selfcomplementarity, Tm and AG (OLIGO computer program, Sequence Analysis Workbench, University of Alabama, Birmingham, USA), and then checked for homology with any other rat mRNA sequences @sru computer program, Daresbury. UK, using the EMBL sequence database). Each ODN showed complete specificity for the target mRNA [3,4]. The uptake of the ODNs was investigated i n H4 rat hepatomacells. These were subcultured i n a % well cell culture cluster (Costar, UK) using Dulbecco's MEM medium (Gibco, UK) with 12% foetal calf serum (FCS. Gibco). The medium was changed to 1% FCS 24 hours later, at which time digoxigenin (dig)-labelled MT-1 or MT-2 ODNs. or fluorescein-labelled M l I (R&D Systems, UK), were added at final concentrations of 2.5 or 10 pM, either in the absence or i n the presence of 5 pg/ml lipofectin (Gibco). Incubation was continued for 18 hours, after which time the ODNs were visualised by either direct detection of the fluorescein-label or by immunological detection of the digoxigenin label. For fluorescein detection, cells were gently rinsed with PBS 18. I mM Na2HP04, 1.9 mM NaH2P04.2H20, 0. ISM NaCI, (pH 7.4)], fixed with 4% paraformaldehyde in PBS for 10 min on ice, washed 5 times with PBS and mounted in Citifluor. For the detection of the dig-labelled ODNs, cells were washed and fixed as above, and then washed twice with PBS and permeabilised for 5-10 min at 4°C with 0.2% Triton in 4% paraformaldehyde in PBS. Cells were washed twice with PBS and twice in buffer 1 1100 mM Tris (pH 7 . 3 , 150 mM NaCI]. To prevent non-specific binding of the antibody, the cells were, at this stage, incubated for 30 min at room temperature with 0.5% w/v blocking reagent (Boehringer) in buffer I . Cells were then briefly washed with buffer 1 and incubated for 45 min at room temperature with 1: IOOdilution of anti-dig-alkaline phosphatase conjugate (antidig-AP from Boehringer) in buffer 1. After another two 15 min washes in buffer 1 they were washed once for 2 min with buffer 2 [ 100 mM Tris (pH 9 . 3 , 100 mM NaCl , SO mM MgC121 and then incubated for 25 min in the dark with 45 pl of NBT (Boehringer) and 35 pl of Xphosphate (Boehringer) in 10 ml of buffer 2. Finally cells were washed in 10 mM Tris (pH 8.0). 1 .O mM EDTA to stop the reaction, washed twice in PBS and photographed.