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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA042354-03
Application #
3183530
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Robinson, D F; Maxwell, I H (1995) Suppression of single and double nonsense mutations introduced into the diphtheria toxin A-chain gene: a potential binary system for toxin gene therapy. Hum Gene Ther 6:137-43
Cook, D R; Maxwell, I H; Glode, L M et al. (1994) Gene therapy for B-cell lymphoma in a SCID mouse model using an immunoglobulin-regulated diphtheria toxin gene delivered by a novel adenovirus-polylysine conjugate. Cancer Biother 9:131-41
Harrison, G S; Long, C J; Maxwell, F et al. (1992) Inhibition of HIV production in cells containing an integrated, HIV-regulated diphtheria toxin A chain gene. AIDS Res Hum Retroviruses 8:39-45
Harrison, G S; Long, C J; Curiel, T J et al. (1992) Inhibition of human immunodeficiency virus-1 production resulting from transduction with a retrovirus containing an HIV-regulated diphtheria toxin A chain gene. Hum Gene Ther 3:461-9
Maxwell, I H; Glode, L M; Maxwell, F (1992) Expression of diphtheria toxin A-chain in mature B-cells: a potential approach to therapy of B-lymphoid malignancy. Leuk Lymphoma 7:457-62
Harrison, G S; Maxwell, F; Long, C J et al. (1991) Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells. Hum Gene Ther 2:53-60
Maxwell, I H; Glode, L M; Maxwell, F (1991) Expression of the diphtheria toxin A-chain coding sequence under the control of promoters and enhancers from immunoglobulin genes as a means of directing toxicity to B-lymphoid cells. Cancer Res 51:4299-304
Maxwell, I H; Brown, J L; Maxwell, F (1991) Inefficiency of expression of luciferase reporter from transfected murine leukaemia proviral DNA may be partially overcome by providing a strong polyadenylation signal. J Gen Virol 72 ( Pt 7):1721-4
Fisher, K S; Maxwell, I H; Murphy, J R et al. (1991) Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences. Infect Immun 59:3562-5
Maxwell, I H; Harrison, G S; Wood, W M et al. (1989) A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques 7:276-80

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