Reduction of c-myc expression by an antisense approach under Cre/IoxP switching induces apoptosis in human liver cancer cells

c-myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mrna were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, Hcc-t, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/IoxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc d id so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-m,vc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death, c-Myc reduction under the Cre/IoxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation. 2001