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Viruses, especially the retroviruses, are "experts" at introducing genes into cells - after all, they produce infections by injecting their own DNA (genes) into cells. A report in the October, 1995 issue of the specialist journal Gene Therapy by a team from the Massachusetts General Hospital in Boston demonstrated that implanting cells which produce a retrovirus vector for a gene into tumors growing in mouse brains was more effective at spreading the gene throughout the tumor than implanting similar cells that had been engineered to produce the gene directly rather than through a virus. Scientists have adapted different viruses as vectors, inserting the desired gene into the virus segment that will enter the target cell.
In many cases a modified bacterial or viral product, or even a totally synthetic genetic construct can enter and modify the cell in a process called transfection. This may require such aids as the use of electrical fields or specific cell membrane-altering substances, or inclusion of the gene or DNA construct in some structure that the tumor cell, but not most normal cells, will take up, just as an ameba engulfs food particles. The vehicles in this latter approach are usually fat particles called liposomes. Finally, naked DNA itself might still achieve a sufficient level of uptake into certain tumors, as discussed by Dr. Hart of St. Thomas's Hospital, London, in the October, 1995 issue of Gene Therapy, but incorporation of the added DNA is less efficient than with viral vectors or other carriers. Most approaches in this area are still far from clinical use, although initial steps in this direction have been taken in the US at the National Cancer Institute and other Cancer Centers. An example of this is a clinical protocol to inject into breast, head and neck, and melanoma tumor sites in patients, their own skin fibroblast cells, genetically modified to produce interleukin-12, a potent biological antitumor substance. This is being done at the Pittsburgh Cancer Institute, as reported at the 2nd European Conference on Gene Therapy of Cancer held in September, 1995.
The November, 1995 issue of the scientific journal Gene Therapy includes abstracts of a series of presentations at a Japanese scientific meeting on gene therapy. There is no intention to imply that similar work is not being undertaken elsewhere, it certainly is and especially in the US, but examples from this journal help illustrate the wide range of approaches that are being taken in this field.
An antisense gene as its name implies is complementary to the target gene sequence, and its presence blocks the tumor gene and knocks out its expression. This approach has stirred much excitement, and the antisense molecule was runner-up for the title "Molecule of the Year" in 1992. However, there is increasing evidence that in this approach, antisense molecules may not always produce their actions by specific antisense interaction with the target molecules, but by other unrelated means instead. An interesting discussion of this may be found in the October 27, 1995, issue of Science.
Whatever might prove to be the critical mechanism of action, a number of studies in Gene Therapy used the antisense approach. In one example, researchers at the Japanese National Cancer Center Research Institute used liposomes containing an antisense gene to a mutant gene (K-ras) in pancreatic cancer cells. In this study the antisense gene inhibited the growth of the pancreatic tumor cells by 83%. A second example was presented by scientists at the Jikei University School of Medicine in Tokyo, who used a combination of radiation therapy with radiolabeled gallium citrate and gene therapy with a synthetic construct against a pancreatic cancer gene to kill the cells.
Esophageal cancer, like pancreatic cancer is very resistant to treatment and therefore an appropriate target for new therapeutic approaches. In a study by researchers from Chiba University, Japan, a retroviral vector was used to transfect three different genes into human esophageal cancer cells. One gene was for the growth factor GM-CSF, which can affect interaction between the tumor cells, another for the herpes virus enzyme thymidine kinase which made the cells sensitive to the drug ganciclovir, and the third was the normal p53 gene (to counteract abnormal tumor p53) which suppressed the growth of the tumor cells.
A similar procedure to sensitize brain tumors to ganciclovir using the herpes virus thymidine kinase was reported by scientists from the Massachusetts General Hospital in Boston at the 2nd European Conference on Gene Therapy at King's College, London, in September. In this study in rats, 89% of the treated animals were free of tumor 109 days after original implantation of tumor, versus only 20% of those who received ganciclovir without gene therapy.
In "War of the Worlds," H. G. Wells had the Martians destroyed by "humble" and primitive bacteria, after advanced human technology had failed miserably. Modern genetic technology is bringing the novel into real life by reaching back to harness an even more primitive life stage, the RNA enzyme or ribozyme, to attack deadly viruses such as HIV, and now tumors also. Ribozymes may be the remnants of a very early period in life's history (the RNA world) when living organisms may have had no genes composed of DNA or enzymes made of protein. RNA, the nucleic acid based on ribose-containing nucleotides, may have served as both gene and enzyme. Ribozymes come in two varieties named for the shapes of their molecules as hairpin and hammerhead. Hammerhead ribozymes are the ones currently being used for their ability to cleave linkages between component nucleotides and so to inhibit expression of specific genes. Specific new ribozymes have been developed from those occurring naturally. Two examples illustrate this.
Acute myeloid leukemias are associated with a gene, AML1/MTG8, made by bringing together (translocation) two normal genes on different chromosomes and fusing them. Scientists from Keio University, the National Defense Medical College, and the Saitama Medical School in Japan developed hammerhead ribozymes specifically targeted to split the RNA product of the AML1/MTG8 fusion gene and prevent it from acting. Another example of ribozyme technology, also from the National Defense Medical College, Japan, used hammerhead ribozymes to overcome drug resistance, a major limitation on chemotherapy. These ribozymes targeted the RNA product of the MDR1 gene, the multi-drug resistance gene, and the cells then became sensitive to the antitumor drug vincristine. The same abstract also described a ribozyme targeting an overexpressed growth factor gene that self- stimulated tumor growth; the ribozyme blocked formation of the growth factor and inhibited cell division. Further studies with these substances should be interesting, and of great potential future importance.
As a final comment on gene therapy, we should point out that since the new gene will not become implanted in every tumor cell, or even in the majority of them, any gene strategy must be able to affect the tumor cells that do not have the introduced gene. This is known as the "bystander effect," and may operate through cell-to-cell contact, through initiating a destructive immune process, or by producing substances that diffuse out to affect neighboring cells. A report from a team at the Massachusetts General Hospital, appearing in the October, 1995 issue of Clinical Cancer Research illustrates the latter process. They transfected a gene for metabolizing the drug cyclophosphamide to its active product into brain tumor cells. Even though only 10% of the cells acquired the gene, the bystander effect led to similar tumor cell kill among both genetically altered and the unaltered cells, since the active metabolite made in the altered cells spread to the unaltered ones also.