Toxic effects are the major drawback of most anticancer therapeutics and are in the top list of patients' worries. Therefore, our goal is to develop technology for cancer treatment that is based on biological mechanisms of action, broadly applicable, and yet minimally toxic so that the treatment will be more effective and less toxic. The goals of the proposed studies are to evaluate the therapeutic and toxic effects of human Bax and human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genes when their expression is driven by the human telomerase reverse transcriptase (hTERT) promoter and to develop better strategies for optimal therapeutic effects. The hypothesis to be tested is that the high tumor-specific expression of the Bax and/or TRAIL genes will eliminate tumor cells but spare normal cells their toxic effects. We demonstrated that direct transfer of the TRAIL or Bax gene resulted in antitumor effects in both p53-sensitive and p53-resistant tumor lines and that hTERT promoter was able to prevent toxic effects of the Bax or TRAIL gene without compromising their antitumor activities. Moreover, single, bicistronic adenoviral vectors expressing the Bax or TRAIL gene from the hTERT promoter were also constructed and their functionality characterized. Preliminary data from studies in progress have shown that these vectors are potentially effective in the treatment of cancers and that they are minimally toxic. Yet, what remains unknown is whether 1). resistance develops to adenovector-mediated proapoptotic gene therapy and, if so, what the possible mechanisms are and the resistance can be overcome the resistance; 2). combining mitochondrion apoptotic genes (such as Bax) with membrane-apoptotic genes (such as TRAIL) improves therapeutic effects, especially in cancers that are resistant to conventional therapy; and 3). hTERT-TRAIL and hTERTBax vectors can be used to treat established metastatic tumors that are resistant to chemotherapy in vivo and what the treatment-related toxic effects are, if any. This proposal is designed to address these unknowns. Success of the proposed studies will lead to new therapeutics for refractory metastatic disease. It will also provide insight into mechanisms of resistance to apoptosis induction in cancer cells.