Ab. Abdelmageed et Kc. Agrawal, ANTISENSE DOWN-REGULATION OF METALLOTHIONEIN INDUCES GROWTH ARREST AND APOPTOSIS IN HUMAN BREAST-CARCINOMA CELLS, Cancer gene therapy, 4(3), 1997, pp. 199-207
The association of increased metallothionein (MT) gene expression in b
reast cancer with metastasis and poor prognosis has led us to investig
ate the hypothesis that inhibition of MT gene expression may elicit an
tiproliferative effects in breast carcinoma MCF7 cells. To monitor the
effect of downregulation of MT protein on growth, MCF7 cells were tra
nsiently transfected by electroporation with an 18-mer MT antisense ph
osphorothioate oligomer (AO) or an 18-mer random oligomer (RO). The MT
-AO is complementary to the region 7 bases downstream from the AUG tra
nslational start site of the hMT-IIA gene. Transfection of MCF7 cells
with the AO inhibited cell growth by 50-60% at 72 hours when compared
to control cells or the cells transfected with RO. The AO-induced grow
th inhibition was associated with alterations in morphology suggestive
of apoptotic cell death. This was further confirmed by DNA linker cle
avage into oligonucleosomal fragments and decreased bcl-2 protein leve
ls in AO-transfected cells as opposed to the RO-transfected cells. Rev
erse transcriptase polymerase chain reaction analysis showed that AO i
nduced a 2-fold increase in the levels of c-fos and p53 transcripts in
comparison to RO which had no significant effect. Conversely, c-myc t
ranscripts were decreased by 2.5-fold in the AO-transfected cells when
compared to the controls. Furthermore, MCF7 cells transfected with an
expression plasmid pBAcNEO-sMT-IIA encompassing human MT-IIA cDNA, co
nstitutively driven by beta-actin promotor, caused a 2.5-fold increase
in intracellular levels of MT, as judged by PCR and western blot anal
ysis, in comparison to the cells transfected with pBAcNEO plasmid. In
contrast to the AO-induced growth inhibition, overexpression of cytopl
asmic MT increased the cell multiplication by 2-fold compared with con
trol cells or the cells transfected with the control plasmid 72 hours
post-transfection. Moreover, the effects of AO on oncogene expression
were reversed on increased expression of MT. These data suggest that o
verexpression of MT potentiates the growth of MCF7 cells, whereas down
regulation of MT elicits antiproliferative effects.