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Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing
Abstract:
As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously 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 such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.
Insights
Researchers explored selective tumor cell killing using introduced toxin genes. Exploiting cell-specific gene regulation, they demonstrated feasibility by expressing diphtheria toxin A-chain (DT-A) in human cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Traditional cancer treatments often lack specificity.
- Targeting tumor cells via surface receptors has limitations.
- Investigating intracellular toxin gene expression offers an alternative strategy.
Purpose of the Study:
- To assess the feasibility of selective tumor cell killing through exogenous toxin gene expression.
- To explore the use of tissue-specific regulatory elements for targeted cell suicide.
- To evaluate the impact of diphtheria toxin A-chain (DT-A) expression on human cell lines.
Main Methods:
- Transfection of human cell lines (HeLa, B-lymphoblastoid, 293) with plasmids encoding the DT-A gene.
- Co-transfection with reporter genes (chloramphenicol acetyltransferase) to assess gene expression.
- Inclusion of enhancer elements in DT-A plasmids to study regulatory effects.
- Selection of G418-resistant transformants to evaluate cell viability.
Main Results:
- DT-A gene expression inhibited reporter gene activity in a cell-dependent manner.
- Enhanced inhibition was observed in 293 cells, which express adenovirus E1A and E1B products.
- The presence of the DT-A sequence prevented the recovery of viable G418-resistant 293 cell transformants.
- Reporter gene expression in B-cells was further reduced by an immunoglobulin enhancer.
Conclusions:
- Cell-specific regulatory mechanisms can be exploited for targeted cancer cell killing.
- Exogenous expression of toxin genes offers a promising approach for selective tumor cell elimination.
- The study demonstrates the potential of gene therapy for inducing cancer cell suicide.