408s
Biochemical Society Transactions ( 1 996) 24
Uptake and effects of antisense oligodeoxynucleotides
specific for rat metallothionein -1 and -2 mRNA in H4
rat hepatoma cells in culture
a
M. HELENA VASCONCEI,OS*, SHUK-CHING TAM#, JOHN
H. BEAITIE *and JOHN E. HESKETH*
*Division of Biochemical Sciences, Rowett Research Institute,
Bucksbum, Aberdeen AB2 9SB, UK; #Department of Chemistry,
University of Aberdeen. Aberdeen AB9 2UE, UK
Metallothionein is an ubiquitous metal-binding protein
which is inducible by various stimuli such a s Cd. Cu, Zn,
dexamethasone or various "stress" factors. In rat liver, there m
two isoforms of metallothionein (MT-I and MT-2) but it 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 13.41were 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, T m 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 p M , 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]. T o
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 2 5 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.
Abbreviations used: FCS (foetal calf serum); dig (digoxigenin);
MT (metallothionein); NBT (4Nitro blue tetrazolium chloride 70%
vlv in DMF); ODN (oligodeoxynucleotide); Xphosphate (S-Bromo4chloro-3-indolyl-phosphate4-toluidine salt solution in DMI-?
C
Figure - lmmunocvtochemical demonstration of the uotake of dik
labelled MT-2 ODN by H4 rat hepatoma cells in the oresence of
limfectin. Control cells (a) were incubated for 18 hours in
Dulbecco's MEM medium with 1% FCS and lipofectin (5 pglml).
Cells treated with 2.5 p M MT-2 ODN without lipofectin show no
uptake of the ODN (b). Cells treated with lipofectin and diglabelled MT-2 ODN [2.SpM (c) or lOpM (d)] show uptake of the
ODN. Bar represents 20 pm.
Results from the dig-labelled ODN uptake studies showed
that the MT-2 ODN was taken up by the H4 rat hepatoma cells. at a
concentration as low as 2.5 pM, in the presence of lipofectin but
not in its absence. The colour developed was more intense with the
10 pM than with the 2.5 p M ODN concentration. suggesting that
the ODN uptake was concentration dependent (see Figure).
However, it was not possible to detect the presence of dig-labelled
MT-I ODN in the cells treated with this ODN. Nevertheless,
further studies with fluorescein-labelled ODN showed that these
cells take up the MT-I ODN, in a concentration dependent manner,
in the presence of lipofectin but not in its absence. We conclude
that both ODNs penetrate the cell membrane in the presence of the
carrier lipofectin, in a concentration dependent manner.
The effect of MT-I ODN treatment on the MT-I content of
these cells was studied using a radioimmunoassay 151. Our
preliminary results show that, compared to cells treated with
lipofectin alone, those treated with the MT-I ODN showed no
reduction in M T - I protein levels. Indeed, the data suggests that
both the M T - I antisense and the sense ODN caused a small
increase (up to 2 fold) in the MT-l protein content. It appears
therefore that these ODNs may cause a non-specific increase in MT
protein content. A similar effect has been seen previously with a
sense ODN targeting the ATG site of insulin-like growth factor I
161.
This work waq supported by the Scottish Oflice Agnculturc. Environment and
Fishenes Departmcnt (SOAEFD). M. Helena Vasconcelos was lundcd by
JNICT (Junta Nacional de Investiga@o Cientifica e Tecnol6gica) from Portugal
and by the British Council. We thank David Brown for making some
oligodeoxynucleotide.
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