"Pol zeta appears to be the only one of a group of specialized DNA polymerases that is critical for development in animals," explained John P. Wittschieben, Ph.D., research instructor in the department of pharmacology, University of Pittsburgh School of Medicine, and first author of the study. "Moreover, its loss in animal cells plays a significant role in the development of chromosomal instability, which is a hallmark of cancer. Therefore, we believe its function may be to suppress the development of tumors."
Although DNA polymerases--enzymes responsible for copying, editing and repairing genes and surrounding DNA--generally have the ability to make completely accurate copies of strands of DNA, in certain situations damaged areas, called lesions, can bring this replication machinery to a complete halt. In the last few years, scientists have learned of the existence of a variety of so-called lesion-replicating polymerases that can overcome these replication "stop signs" and keep cells dividing that would otherwise be killed off by their own suicide mechanisms.
First discovered in budding yeast cells, and later in plants and animals, pol zeta has the remarkable ability, in the test tube, to efficiently extend DNA with lesions that stop most other DNA polymerases in their tracks. Other research has shown that inactivation of this lesion-replicating enzyme in yeast leads to a dramatic decrease in the frequency of mutations induced by a wide range of DNA damaging agents.
In this study, Dr. Wittschieben--working in the laboratory of Richard D. Wood, Ph.D., professor of pharmacology, the Richard M. Cyert Chair in Molecular Oncology and director of the molecular and cellular oncology program at the University of Pittsburgh Cancer Institute--sought to determine pol zeta's key role in mice cells. To do this, Drs. Wittschieben and Wood disabled, or "knocked out," the gene for pol zeta's Rev3L subunit, the part with the lesion-replicating capabilities. However, knocking out the Rev3L gene proved lethal to the mice embryos. The investigators nevertheless isolated fibroblasts from these embryos to see if they could be kept alive in culture. After repeated attempts, the mouse embryonic fibroblasts, or MEFs, failed to divide and died within a few weeks or months.
Suspecting that the MEFs were dying because they were self-destructing, or undergoing apoptosis, the investigators then knocked out the gene for a protein known as p53, which is a cell-suicide-signaling molecule. After matings between the p53 knockout mice and Rev3L knockout mice, the investigators isolated and cultured MEFs from all the offspring of the matings to see if any would grow. Unfortunately, the cells all failed to divide. However, three months later, some cells began to grow and at a surprisingly robust rate.
"Once the cells in which Rev3L and p53 had been knocked out began to divide, they did so very rapidly," said Dr. Wittschieben. "Because the only Rev3L-deficient cells that began dividing also were p53 deficient, we believe that knocking out their apoptotic mechanism was key to this viability. However, they didn't begin dividing right away, so something else must have happened. We are still not sure what that something else is."
When the investigators looked for evidence that these cells were different from normal cells, they didn't have to look far. Examination of the cells' chromosomes showed not only a dramatic ten-fold increase in the incidence of swapping and fusing of genes and other genetic material between chromosomes, but also an increase in the number of chromosomes compared to normal cells.
The high frequency of DNA rearrangements in Rev3L/p53-deficient cells suggests that pol zeta in normal cells is responsible for preventing double-stranded breaks from occurring in chromosomes. When pol zeta is absent, it leads to a massive amount of double stranded breaks, some of which are repaired correctly and others that are repaired incorrectly by being fused to other genes or chromosomes.
According to Dr. Wood, these findings have significant implications for human cancer research, in that such a high degree of chromosomal instability is a characteristic of cancer cells. Furthermore, the human Rev3L gene is located in a segment of chromosome 6 where multiple tumor suppressor genes are believed to reside and a slew of human cancers, including a number of leukemias and lymphomas, are associated with chromosomal instabilities in this particular region of chromosome 6.
"Although it requires further investigation, we believe that mutations in this part of chromosome 6 could occur during the development of some cancers and this may have prognostic and therapeutic implications. We are now investigating this hypothesis by selectively deleting the Rev3L gene in adult mouse cells to study how the loss of DNA polymerase zeta influences the development and progression of spontaneous cancers," explained Dr. Wood.
This work was supported by a grant from the National Cancer Institute, National Institutes of Health to Dr. Wood. In addition to Drs. Wittschieben and Wood, others involved in this study include Shalini C. Reshmi, Ph.D., and Susan M. Gollin, Ph.D., University of Pittsburgh Graduate School of Public Health.