Overview
Quantitation of Radiation Induced Deletion and Recombination Events Associated with Repeated DNA Sequences
Principal Investigator:
Richard R. Sinden, Ph.D.
Organization:
Texas A&M University - Institute of Biosciences and Technology
Technical Summary
Manned exploration of space exposes the explorers to a complex and novel radiation environment. The galactic cosmic ray and trapped belt radiation (predominantly proton) components of this environment are relatively constant, and the variations with the solar cycle are well understood and predictable. The level of radiation encountered in low earth orbits is determined by several factors, including altitude, inclination of orbit with respect to the equator, and spacecraft shielding. At higher altitudes, and on a Mars mission, the level of radiation exposure will increase significantly. A significant fraction of the dose may be delivered by solar particle events which vary dramatically in dose rate and incident particle spectrum. High-LET radiation is of particular concern. High-LET radiation, a component of galactic cosmic rays (GCR), is comprised of a variety of charged particles of various energies (10 MeV n-1 to 10 Gev n-1), including about 87 percent photons, 12 percent helium ions, and heavy ions (including iron).
These high energy particles can cause significant damage to target cells. The different particle types and energies result in different patterns of energy deposition at the molecular and cellular level in a primary target cell. They can also cause significant damage to other, nearby cells as a result of secondary particles. Protons, for instance produce secondaries that include photons, neutrons, pions, heavy particles, as well as gamma rays. Heavy ions deposit energy in a "track" in which the magnitude of the damage varies as the particle loses energy. Heavy ions produce secondary delta rays, or electrons. The distribution of damage through tissue is described by a Bragg curve which will be characteristic for different energies. Needless to say there are differences in the RBE of protons and α particles.
High-LET heavy ions are particularly damaging to cells as they do continual damage throughout their track. Differences in these energy deposition patterns can significantly influence the nature of DNA damage and the ability of cellular systems to repair such damage. It has been suspected that these differences also affect the spatial distribution of damage within the DNA of the interphase cell nucleus and produce corresponding differences in endpoints related to health effects. The interaction of a single high-LET particle with chromatin has been suggested to cause multiple double strand breaks within a relatively short distance. In part this is due to the organization of DNA into chromatin fibers in which distant regions of the DNA helix can be physically juxtaposed by the various levels of coiling of the DNA. This prediction was confirmed by the detection of the generation of double strand DNA fragments of 100-2000 bp following exposure to high-LET ions (including iron).
While it is very clear that ionizing radiation can cause cytogenetic damage and cancer, relatively little is yet known about the mutagenic or carcinogenic effects of high energy HZE particles in cells. High-LET radiation produces proportionally more double-strand than single strand breaks compared with low-LET radiation. Double-strand breaks are likely responsible for the cytogenetic damage visible as chromosomal aberrations, transformation, mutations, and delayed cell death.
Nearly one-third of the human genome is composed of DNA repeats, which include simple mono-, di-, tri-, and tetranucleotide repeats; widely separated small and large repeats; and inverted repeats. Mutations associated with repetitive DNA are a source of many genetic diseases and cancer. Therefore, understanding how the various kinds of repeats contribute to the disease burden and understanding the impact of DNA damage on repeat-associated genomic instability is important for human health. Such repeated DNA sequences are likely to be very prone to mutation following exposure to high-Z high-energy (HZE) particles during space flight. Cells in the direct line of the HZE particle sustain a high dose of energy while cells surrounding the primary tract sustain a lower dose of energy from the energetic delta rays (electrons) produced by HZE particles. Therefore, the nature and pattern DNA damage to cells in tissue upon irradiation with HZE particles is particularly complex. It is important to understand the types of mutational changes induced by both the HZE particles as well as the delta rays.
Given the high frequency of occurrence of repeated DNA sequences it is highly likely breaks or base damage from radiation will occur within these sequences. Moreover certain processes of repair and recombination involve the generation of free 3' ends in DNA and extended single-strand regions that expose repeats to recombination or primer template misalignments. Therefore, the molecular events that are responsible for cytogenetic damage (chromosomal breaks and rearrangements) and other mutations (point mutations, frameshifts, small deletions and duplications) in many cases will involve primer template misalignments. (Note that we have shown that the molecular mechanism for a hotspot for several +1 frameshift mutations involves intermolecular strand switch events (primer template misalignments) that occur specifically in during leading strand replication at a region containing DNA repeats (Rosche et al., 1998).) It is also possible that a cell sustaining substantial damage from a heavy iron particle hit may saturate some or all of its repair capability, or induce an error prone mode of repair to mediate survival. The types of assays we are developing will provide sensitive reporters of the replication/repair fidelity of a cell following damage from HZE particles. It is the fidelity of this process which, if compromised, will ultimately lead to carcinogenesis or other detrimental effects of radiation damage. With the sensitive reporter constructs we are developing, the protective effects of chemopreventive measures or countermeasures can be quickly established. A major goal of this project was to provide a rapid way to test the efficacy of various countermeasures and chemopreventive drugs with respect to mutation minimization and cancer prevention. The sensitive, relative rapid assay being developed here would compliment the long-term Dicello rat study being conducted at Johns Hopkins.
Goal
The goal of this proposal was to develop data on the relationship between gene mutations, including deletions and recombination associated with direct repeats, and the quantity and quality of the radiation that interacts with the biological system so that countermeasures designed to minimize the health risks of radiation exposure in space can be devised. This goal could be accomplished by quantifying the rate of deletions between direct repeats, which may involve primer-template misalignment, recombination, or gene conversion in human cells following exposure to radiations which reproduce the energy deposition patterns produced in individual cells by the radiation environment in space. Using cell lines that provide sensitive reporters of mutations involving deletions between direct repeats and recombination events, we measured the rate of mutations in irradiated cells and in progeny of irradiated cells, following exposure to high energy alpha particles. We also planned to analyze several biological endpoints in other cells lines that do not contain genetically selectable end points, but which contain long tracts of direct repeats (1.8 mb) or inverted repeats (15.3 kb). In addition, we also began developing additional reporter constructs for application in Sprague-Dawley rat mammary cells (and eventually rate) to increase the sensitivity of measuring deletions and recombination events mediated by DNA repeats. This will complement the long-term rat carcinogenesis study of the Radiation Effects Group, by providing a rapid, sensitive screen for the effects of chemopreventive and radioprotective drugs on genome instability following exposure to HZE particles and protons.
Hypothesis
The hypothesis driving this proposal is that DNA damage introduced by high-energy (HZE) particles induces aberrant DNA repair events, involving repeated DNA sequences that lead to recombination, gene conversion, or other mutation, that initiate the sequence of cytogenetic and functional changes which manifest themselves as the long term health effects of radiation exposure in space, including cancer. Knowing the types of mutational events induced by different radiations will contribute to sound decisions for optimizing shielding and reducing biological consequences through use of radioprotective drugs or various countermeasures. The cell lines and procedures utilized in this proposal will be useful for testing the efficacy of various countermeasures and chemopreventive drugs.
Key Findings
The survival of four reporter cell lines (122-2, F14C-23, 7#7-7, and 3134) following exposure to 250 KeV X-rays has been measured. All cell lines were sensitive to X-rays in a range that would allow them to be used as reporter cell lines for radiation damage.
- The frequency of deletion of an inverted repeat and a nonpalindromic sequence from a neo gene in human 122-2 and F14C-23 cells have been measured (by isolating clones resistant to the antibiotic G-418) following exposure to 250 KeV X-rays.
- The nature of the reversion events, which involve precise deletions between direct repeats, has been analyzed by PCR analysis.
- The rate of reversion or the mutant frequency for the deletion mutations in the neo gene have been calculated for control and X-ray exposed cells. The frequencies are about 1-2 x 10-7 in sham (non irradiated) cells. The rate increases by as much as a factor of 60 following exposure to X-rays.
- The survival and G-418 reversion frequencies following exposure to 1000 MeV Fe particles at the BNL4 and BNL5 runs have been measured. Following Fe exposure, the rate of G-418 reversion increases as much as a factor of 100.
- We have successfully cloned a 770 bp perfect inverted repeat (2 x 385 bp Alu sequence) and a 763 bp inverted repeat with a 39 bp nonpalindromic center (2 x 362 bp + 39 bp) in E. coli. This has taken considerable effort as this is six to sevent times longer than any inverted repeat we have previously cloned. A number of modifications to existing protocols had to be developed to get this. We are putting SphI adaptors on it to clone it into the neo gene in pJJ999 (the vector be used for electroporation into rat cells.
Unfortunately, the project has come to an end just as we were at the verge of obtaining new reporter constructs (with the alu inverted repeats) that should work quite well for these experiments. More research and development was necessary in this area that was originally anticipated. The constraint of minimal personnel slowed progress in this area.
Implications of Findings
This project was directed toward understanding the molecular mechanisms of radiation damage and repair in cells. These studies were designed to determine the relationship between the energy deposition pattern of radiation and its ability to increase the frequency of specific mutation events. DNA repeats are involved in many deletions and rearrangements associated with human disease. The reporter constructs, cell lines, and procedures to have been developed in this proposal will be directly applicable to studies on the effect of radioprotective drugs, as they would provide a very sensitive and relatively rapid quantitative assay for their effects. Moreover, these reporter constructs can be introduced into other types of cells and transgenic animals, including the Sprague Dawley rat. Thus, integration into the Dicello rat study may provide a way for ascertaining the effects of radiation and the protective effects of countermeasures, in a fraction of the time required for a long-term rat study. (Note, however, that the rat study must be completed for integration with a more rapid screen for countermeasures.)
Eventually our results will be useful for understanding the genetic predisposition to disease and cancer from radiation, which will be important for the potential genetic screening of astronauts.
Many other questions can be addressed with further application of our system, including those related to: chemical and biological agents that might be implemented to mitigate acute exposures, efficacy of radioprotectants, questions of shielding effectiveness, questions of fluence and fluence rate effects, and efficacy of nutritional supplements.