EXPRESSION AND LOCATION OF PRO-APOPTOTIC BCL-2 FAMILY PROTEIN BAD IN NORMAL HUMAN TISSUES AND TUMOR-CELL LINES

The BAD protein is a pro-apoptotic member of the Bcl-2 family whose ab ility to heterodimerize with survival proteins such as Bcl-X-L and to promote cell death is inhibited by phosphorylation. Monoclonal antibod ies were generated against the human BAD protein and used to evaluate its expression by immunoblotting and immunohistochemistry in normal hu man tissues and by immunoblot analysis of the National Cancer Institut e anti-cancer drug screening panel of 60 human tumor cell lines. BAD p rotein was detectable by immunoblotting in many normal tissues, with t estis, breast, colon, and spleen being among those with the highest st eady-state levels. Immunostaining of tissues revealed many examples of cell-type-specific expression of BAD, suggesting dynamic regulation o f BAD protein levels in vivo, In many types of normal cells, BAD immun oreactivity was associated with cytosolic organelles resembling mitoch ondria, suggesting that BAD is often heterodimerized with other Bcl-2 family proteins in vivo, The relative levels of BAD protein varied wid ely among established human tumor cell lines, with colon, lung, and me lanomas generally having the highest expression. As a group, hematopoi etic and lymphoid lines contained the least BAD protein. The BAD prote in derived from 11 of 41 tumor Lines that expressed this pro-apoptotic protein migrated in gels as a clear doublet, consistent with the pres ence of hyperphosphorylated BAD protein, Taken together, these finding s define for the fit st time the normal cell-type-specific patterns of expression and intracellular locations of the BAD protein in vivo and provide insights into the regulation of this pro-apoptotic Bcl-2 fami ly protein in human tumors.