Tumor Selectivity in an Adenoviral Vector: Are we Clever or What?
ABSTRACT & COMMENTARY
Synopsis: Many tumors have defects that interfere with normal G1 cell cycle arrest. This may lead to overexpression of genes normally inhibited by the G1 checkpoint arrest proteins. An adenoviral vector containing an E2F-responsive promoter was shown to be preferentially expressed in tumor cells in vivo.
Source: Parr MJ, et al. Nature Med 1997;3:1145-1149.
The cell cycle is designed with several checkpoints at which the cell takes stock of how it is functioning and decides whether it can proceed with the next step in the cycle. The G1 checkpoint seems to be designed to assess whether the genome is sufficiently intact, and whether it is safe to proceed with the duplication of the genetic material in S phase. There is another checkpoint in G2 that takes inventory of the amount of DNA in the cell to be sure that a tetraploid number of chromosomes are present so that mitosis may proceed safely.
Nearly every human tumor has genetic alterations that interfere with the function of the G1 checkpoint. More than half of human tumors have mutations in p53, and most of the rest have mutations that disrupt the retinoblastoma protein (pRB) function, either mutations in the gene for pRB or mutations in upstream pRB regulators such as cyclin D1 or the cyclin-dependent kinase inhibitors, such as p16. A prediction of these data is that there should be an abundance of transcription going on in the cell driven by E2F promoters because the critical function of pRB is to repress E2F activity. No pRB function should mean lots of E2F activity.
Parr and colleagues sought to test this prediction by construction of an adenoviral vector containing a gene (e.g., beta-galactosidase) under the influence of an E2F promoter. That is, it could only be expressed if it was activated by E2F binding. They administered the vector to tumor-bearing animals and found that the introduced gene was expressed in tumor cells but not in normal cells. They replaced the beta-galactosidase gene with the gene for herpes simplex virus thymidine kinase, a gene which, if expressed, should make the expressing cell susceptible to killing by ganciclovir. They found that more than 75% of animals treated with ganciclovir after administration of the adenoviral vector containing thymidine kinase under the control of an E2F promoter were cured. The appropriate controls found no effect from ganciclovir in the absence of thymidine kinase expression.
COMMENTARY
Our efforts to design tumor-specific therapy have generally relied on targeting cells that were proliferating. However, many normal tissues that are renewable, especially the bone marrow and gastrointestinal tract, have normal cells that are affected by proliferation-specific interventions. It is certainly possible that the trick reported here will be no more selective than cytosine arabinoside, but at least it reflects a level of thought that is more sophisticated.
At the G1 checkpoint, pRB binds to E2F family transcription factors and keeps them from translocating to the nucleus and turning on genes with E2F promoters. Such genes include DNA polymerases and other enzymes and proteins needed for DNA replication. When pRB is phosphorylated, it lets go of E2F and DNA synthesis (S phase) proceeds. Mutations that interfere with the pRB function are common in cancer cells. This should mean that E2F transcription factors are free to activate the production of all the genes whose transcription is under the control of an E2F promoter. This group has reasoned that, if that is true, maybe they can add a gene to the cell under the influence of that promoter that will get the cell killed.
Adenoviral vectors are attractive candidates for gene therapy because they infect many of the cells with which they come in contact. The strategy devised by Parr and colleagues minimizes the risk of having adenovirus infecting cells that are normal and not neoplastic, because in normal cells, the thymidine kinase gene will have trouble getting expressed because all of the E2F factors are bound to pRB. Only cells without pRB (and we hope these are all cancer cells) will express the gene and only those cells will become susceptible to ganciclovir killing.
It is likely that there is something wrong with this idea. We know that only a small fraction of solid tumor cells in patients with cancer are progressing through the cell cycle. What will be the effect of this adenoviral vector on cells that are resting? Such questions cannot be addressed in animal tumor models because all the tumor cells are growing in animal models. Thus, only human clinical trials will be able to address the next questions regarding the efficacy of this clever strategy.
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