A bcl-xS adenovirus selectively induces apoptosis in transformed cells compared to normal mammary cells

Abstract

Oncogenes which drive the cell cycle, such as c-myc, can sensitize cells to apoptosis. This suggests the possibility that the expression of genes such as bcl-2 or bcl-xL is required to inhibit apoptosis induced by oncogene expression. We hypothesized that inhibition of Bcl-2/Bcl-xL by the pro-apoptotic Bcl-xS protein, would result in selective induction of apoptosis in mammary carcinoma cells compared to their nontransformed counterparts. Therefore, we compared the effects of Bcl-xS expression delivered by a bcl-xS adenovirus (bcl-xS-Adv) vector, on viability and apoptosis of nontransformed versus transformed mammary epithelial cells. We report that c-myc-transformed murine mammary cells are extremely sensitive to apoptosis induced by the bcl-xS adenovirus (bcl-xS-Adv) vector, whereas immortalized, nontransformed murine mammary cells are relatively resistant to apoptosis induced by this vector. Likewise, human mammary epithelial cells transduced with c-erbB-2 were more sensitive to apoptosis induced by the bcl-xS vector than the nontransformed parental cells. Similar results were obtained when we tested the effects of bcl-xS adenoviral infection on primary normal human mammary epithelial cells and SUM-190 PT cells, (a c-erbB-2 over-expressing human mammary carcinoma cell line) grown on Matrigel. These data are consistent with the hypothesis that inhibition of Bcl-2/Bcl-xL can result in selective killing of cancer cells compared to their nontransformed counterparts.

Figure 1

Bcl-xS expression in cells infected with the bcl-xS adenovirus. All cell lines were infected with the bcl-xS or lacZ adenoviral vectors (MOI of 1000). Two days after infection, lysates were prepared and electrophoresed on 12% SDS-PAGE gels. The western blot was probed with antibody to human Bcl-x. The Bcl-xS protein is indicated by the arrow. The heavy band above Bcl-xS represents Bcl-xL, which is also recognised by this antibody. (A) Comma-1D and Myc-83 cells: Lanes 1, 2, and 3 represent lysates of uninfected Comma-1D cells, Comma-1D cells infected with bcl-xS or lacZ viruses respectively. Lanes 4, 5, and 6 represent lysates of uninfected Myc-83 cells, Myc-83 cells infected with the bcl-xS and lacZ adenoviruses respectively. (B) H16N2-PTP and H16N2-c-erbB-2 cells: Lanes 1, 2, and 3 show Bcl-xS expression in uninfected H16N2-PTP cells and H16N2-PTP cells infected with the bcl-xS and lacZ viruses respectively. Lane 4 is blank. Lanes 5, 6, and 7 represent lysates of uninfected H16N2-c-erbB-2 cells, and H16N2-c-erbB-2 cells infected with the bcl-xS and lacZ vectors respectively.

Figure 2

Effect of bcl-xS adenovirus infection on viability of Comma-1D and Myc-83 cells: Each cell type was infected with the bcl-xS or lacZ vectors (MOI's of 50–1000). For each cell type and viral titer, the number of viable cells in bcl-xS-infected samples is expressed as a percentage of total cell number in the lacZ-infected counterpart. The graph shows the viability of the Myc 83 cells (black bars) and Comma 1D cells (white bars) on day 3 after infection with the viral vectors. A statistically significant difference in viability of bcl-xS-infected Myc 83 cells relative to the bcl-xS-infected Comma 1-D cells was observed at MOI's of 250, 500, and 1000 (n=4, P=.005, n=4, p=.022, and n=4, p=.001, respectively).

Figure 3

Effect of Bcl-xS overexpression on viability of H16N2-PTP and H16N2-c-erbB-2 cells. For each cell type and viral titer, the number of viable cells in bcl-xS-infected samples is expressed as a percentage of total cell number in the lacZ-infected counterpart. The graph shows viability of the H16N2-c-erbB-2 (black bars) and H16N2-PTP cells (white bars) infected with the bcl-xS or lacZ vector on day 5 after infection. Comparison of the differences in viability of the bcl-xS-infected c-erbB-2 transductants relative to bcl-xS-infected parental H16N2-PTP cells showed statistical significance at MOI's of 250, 500 and 1000 (n=4, p=.027, n=4, p=.005, and n=4, p=.002 respectively).

Figure 4

Effects of bcl-xS adenoviral infection on normal human mammary cells grown on Matrigel. On day 12 after infection with the viral vectors (1000 MOI), colonies of normal cells were vital stained and photographed at a magnification of 100x. The lacZ-infected, bcl-xS-infected, and uninfected nominal cells are shown in panels A to C respectively. Panel D shows Adv-lacZ-infected normal cells stained for β-galactosidase activity.

Figure 5

Effects of bcl-xS adenoviral infection on SUM-190 PT tumor cells grown on Matrigel. On day 12 after infection with the viral vectors (1000 MOI), colonies of cells SUM-190 PT tumor cells were vital stained and photographed at a magnification of 100x. The lacZ-infected, bcl-xS-infected, and uninfected normal cells are shown in panels A to C respectively. Panel D shows X-gal staining of lacZ-infected SUM-190 PT tumor cells.