Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUCI and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly I I times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the tarnsfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 505-511, 2010