We are exploring the possible killing of tumor cells by expression of an introduced toxin gene (i.e. cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of this approach we transfected human cell lines (HeLa, 293 and B-lymphoblastoid) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. DT-A substantially lowered both stable transformation frequencies and transient expression of chloramphenicol acetyltransferase from a co-transfected plasmid, pSV2cat. This expression level in B-cells was further diminished when the DT-A plasmid contained an immunoglobulin (Ig) enhancer. We shall attempt to maximize preferential toxicity for B-cells as a model for optimizing the toxin gene system. Plasmids will be constructed containing DT-A, together with Ig heavy and kappa chain promoters and enhancers in various combinations. DT-A expression from these constructs will be assessed at the level of DT-A mRNA (single strand nuclease probe protection assay) and functional toxin (estimated by inhibitory effects in transient co-transfection and stable transformation assays.) Expression will be studied following transfection of myeloma cells, B-cells, and pre-B-cells (70Z/3) with or without lipopolysaccharide induction of endogenous kappa gene expression. Constructs giving efficient expression in B-lymphoid cells will be tested in other hematopoietic cells (myeloid and T-cell lines) to determine whether Ig promoters and enhancers show significant activity in these cells. In order to minimize toxicity in non-target cells due to basal transcription of DT-A, we shall explore use of the attentuated tox 176 A chain coding sequence in our expression plasmids and of DT-A anti-sense RNA, transcribed from an opposing downstream promoter. We shall investigate human T-cell lymphotropic virus (HTLV) trans-activation as an additional system for directing cell-specific expression and toxicity of DT-A. Using a plasmid containing the DT-A coding sequence attached to the HTLV II LTR, we hope to observe toxicity restricted to cells expressing the corresponding trans-activator. These studies could lead to therapeutic agents for adult T-cell leukemia and AIDS. This proposal will provide a basis for the subsequent design of efficient viral delivery vehicles directing tissue-specific expression of a transduced toxin gene, in addition to elucidating the role of promoters and enhancers in the tissue-specificity of Ig gene transcription.
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