Brian H. Ladle and Dr. Kim L. O’Neill, Microbiology

As researchers continue to discover the molecular origins of cancer, gene therapy targeting cellular processes gone awry shows great promise as the future in cancer treatment. Extensive research is being done to develop more effective techniques targeting different cellular events. One pathway receiving increased attention is apoptosis (the default mechanisms activated intracellularly by extensive damage, causing the cell to kill itself). Often cancer will develop when genetic mutations or other cellular events render the cells unable to die by apoptosis. My research investigates a novel gene therapy that will restore the cancer cell’s ability to die by apoptosis, while limiting its expression and harmful effects in normal tissue.

The two components of the apoptotic pathway targeted by this therapy are the proteins Bcl-2 and Bax. Past research has shown that the intracellular ratio between these two proteins determines the fate of the cell. High levels of Bcl-2 protect the cell from dying by apoptosis, while high levels of Bax permit the signals to be propagated that will result in cell death (4). Analysis of many cancers reveals either abnormally high levels of Bcl-2 or low levels of Bax (1). Through gene therapy, Bax can be over-expressed and Bcl-2 suppressed, allowing normal cellular mechanisms to kill the cancer cells.

One of the difficulties that must be overcome before a gene therapy can become clinically useful is its lack of specificity. It has proven very difficult to introduce the engineered DNA into a host without also infecting normal tissue. The need exists to discover a method of controlling the expression of the gene therapy to make it cancer-specific. Research by Dr. O’Neill and others indicates that the promoter region (DNA sequence that controls the expression of a gene) of the Thymidine Kinase (TK) gene might render a solution (3). It has been shown that the promoter is highly regula ted and controlled in normal tissue, but in cancer cells it is poorly regulated and very active (2). Thus, placing the gene therapy under the control of the TK promoter will limit its expression to cancer cells, increasing significantly its specificity.

Using standard molecular biology techniques, I have engineered five expression vectors (small pieces of DNA) that will be used to complete this study. The pOP13CAT LacSwitchTM Inducible Mammalian Expression System from Stratagene and the pCR-Blunt-IITOPO vector from Invitrogen were engineered to contain the desired sequences. One vector, pOP13hTKpCAT, contains the Chloramphenicol Acetyl Transferase (CAT) gene under the control of the human Thymidine Kinase promoter (hTKp). This vector will allow for the measurement of the activity of the hTK promoter in various cancer cell lines. The second vector, pOP13RSVBax, contains the bax gene under control of the viral RSV-LTR promoter. The RSVLTR promoter is known to be very active in any cell line and will allow for over-expression of Bax. This vector will be used as a positive control for the experiment. The third vector, pOP13hTKpBax (Fig. 1), is a combination of the first two. The bax gene is under the control of the hTK promoter. This vector will allow for the study of the cancer-specific expression of the TK promoter to over-express Bax. The other vectors contain antisense bcl-2 sequences. Expression of these antisense sequences results in the suppression of Bcl-2 protein levels. The most effecient inhibitory sequence will be determined and used in further studies.

Following the construction of these expression vectors, I have transfected them into a breast cancer cell line. The cells containing the recombinant DNA are isolated by adding antibiotics to the media in which they grow. The vectors contain a gene that confers resistance to antibiotics, so only the cells that contain the vector survive. This process took several weeks. I am now isolating cells that contain the DNA I have engineered. Over the next several months, I will be studying the expression of these vectors by determining the effects on cell viability and measuring the protein levels of Bax and Bcl-2. I plan to submit my results for presentation at the 1999 Annual Meeting of the American Association of Cancer Research.

The results of this research should yield two important advances. If the hTK promoter provides specificity to this gene therapy, the implications will be widespread. This would be a significant step in developing gene therapies that will only be expressed in cancer cells. The other important aspect of this project will be if the combination of Bax over-expression and Bcl-2 suppression yields a more potent therapy than either one separately. If successful it will open up many new avenues of research. This research is pioneering efforts to combine gene therapies. Overall, this project should be an important contribution to the development of safer, more effective gene therapies (5).

References

1. Bargou, R.C., Wagener, C., Bommert, K., Mapara, M.Y., Daniel, P.T., Arnold, W., Dietel, M., Guski, H., Feller, A., Royer, H.D., Dorken, B. Overexpression of the Death- promoting Gene bax- Which Is Downregulated in Breast Cancer Restores Sensitivity to Different Apoptotic Stimuli and Reduces Tumor Growth in SCID Mice. Journal of Clinical Investigation, 97(11):2651-2659, 1996.
2. Iito M., Conrad S.E. Independent Regulation of Thymidine Kinase mRNA and Enzyme Levels in Serumstimulated Cells. Proceedings of the National Academy of Sciences, 6954-6960, 1989.
3. O’Neill, K. L., Grigsby, R.V., Fairbairn, D.W. Thymidine Kinase: The Future in Breast Cancer Prognosis. The Breast, 4:79-83, 1995.
4. Zha, H., Aime-Sempe C., Sato, T., Reed, J.C. Proapoptotic Protein Bax Heterodimerizes with Bcl-2 and Homodimerizes with Bax via a Novel Domain (BH3) Distinct from BH1 and BH2. Journal of Biological Chemistry 271:7440-7444, 1996.
5. I must especially acknowledge the BYU Cancer Research Center, by whom I was funded as a research fellow while performing this research. Also I acknowledge the tremendous support my faculty mentor, Dr. O’Neill, has been in all of my research efforts.