Project 3: Chromosomal changes, genomic instability and the bystander effect

Project Leader: Charles R. Geard

Overiew
Non-targeted radiation responses refer to those phenomena which express in cells or tissues that were not directly hit by ionizing radiation. Such responses are in direct conflict with the long established expectation that only cells whose nuclear DNA has been directly damaged by radiation can express a deleterious response. Clearly, simple extrapolations of declining radiation responsiveness at progressively lower doses may not be tenable in such a scenario. Hence defining the validity, breadth of relevance and mechanistic bases for non-targeted radiation responses is necessary for confidence to be sustained in radiation protection standards. The bystander effect describes the ability of cells targeted and affected by an agent to signal non-targeted cells to initiate and express similar changes. Genomic instability refers to a latent delayed expression of genomic damage in cells multiple generations after response to an initial dose of radiation. Determining the relationship between these two phenomena is a principal thrust of this project, which relies on continuing interactions with Project 1 and Project 2.

Research Aims

Principal hypothesis:
The bystander response and genomic instability have mechanisms in common.
Observation: Both irradiated cells surviving alpha particle irradiation and non-hit, non-contacting bystander cells show similar levels of genomic instability in the form of chromatid aberrations over many generations.

Testable hypothesis:
1. Genomic instability in bystander cells is independent of initial cell-cell contact.
Observations: Chromatid break frequency in G2-PCC`s provides a sentinel monitor of bystander responsiveness. Microbeam irradiation and discrimination protocols allow definitive assessment of known hit cells relative to known non-hit [bystander] cells. The bystander response is independent of the dose or particle fluence delivered to the hit cells.

2. Both bystander responses and subsequent genomic instability depend on initial radiation damage, and share response induction/propagation pathways.
Observations: Site specific microbeam irradiation of extra-nuclear [cytoplasmic] areas of A_L cells results in a maximal 3-fold increase in mutation and only a 20% decline in clonogenicity. This response is abrogated by treatment with DMSO, supporting an oxygen-based free radical origin for this response. That is, DNA in the cell nucleus is not the only target for ionizing radiation responses. Cytoplasmic irradiation of only 20 % of cells also results in a higher mutant yield than expected. That is non-nuclear irradiation can result in initial nuclear changes and can initiate a bystander effect. These results could only have been obtained with a microbeam.

3. Genomic instability manifest following ionizing radiation exposure is not dependent on direct damage to the cell nucleus.
Observations: An awareness of the potential of 3-D tissue culture has been slowly growing. Certainly such systems are closer to the in vivo situation, with cell- cell contacts and extra-cellular matrix interactions. Charged particles in microbeam delivery mode provide a powerful means of inducing damage in defined tissue sites, allowing comparison between bystander responsiveness in cells in 2-D monolayers and cells of the same tissue origin in organized 3-D tissue like models. Both 2-D and 3-D systems have shown responses following microbeam irradiation.

4. Cells irradiated in 2-D monolayers will show similar bystander responses to cells irradiated in 3-D tissue-like constructs.

Research Highlights

1. The RARAF microbeam along with a specific cell discrimination protocol was used to determine the molecular responsiveness of individual hit cells relative to known non-hit bystander cells. A population of normal human fibroblasts was divided in two and half the cells treated with the vital nuclear blue fluorescing dye, Hoechst 33342, while the other half were treated with the vital cytoplasmic orange fluorescing dye, CellTracker Orange. Cells were then mixed in a ratio of 1:1 and plated in microbeam dishes. Cells were plated at a density leading to the majority being distinctly separated from each other. The numbers and co-ordinates of all blue fluorescing nuclei were then recorded prior to site specific nuclear irradiation with defined numbers of alpha particles. Following microbeam irradiation each dish of cells for each particle fluence was placed on an inverted fluorescence microscope, subjected to gentle trypsinization to loosen cell attachment and individual cells expressing blue fluorescence [hit cells] or orange fluorescence [bystander cells] were removed via a capillary pipette attached to a Narashige micro-manipulator, and placed into a PCR micro-centrifuge tube. The amount of the cyclin-dependent kinase inhibitor CDKN1a [p21/Cip1/WAF1] was determined at the mRNA level in individual cells by RT-PCR analysis. Some representative results comparing hit and bystander cell responses relative to similarly handled sham irradiated control cells at one hour post-irradiation are shown. Hit cells, and non-hit cells show clear increases in the levels of CDKN1a mRNA; a definitive demonstration of a bystander effect.

2. The forced condensation of chromosomes in interphase cells by the use of agents such as calyculin, allows for cell cycle stage specific assessment of induced changes in the prematurely condensed chromosomes [PCC`s]. The human-hamster hybrid cell line Al is a mainstay of Project 2, in which mutations in the human chromosome 11 are quantified and characterized, in hit and bystander cells, in the presence and absence of specific inhibitors of members of responsible pathways. Changes in the human chromosome 11 can also be characterized by m-BAND fluorescence in situ hybridization, as shown in the Figure above of PCC`s from different stages of the cell cycle. The fragmentation of chromosomes in S phase cells with ongoing synthesis of DNA precludes assessments of changes in such cells. However appropriate treatments can provide both scoreable G1 and G2 phase PCC`s. Chromosome specific changes are being recorded in cells after site specific nuclear and cytoplasmic irradiation and in bystander cells from these scenarios, along with assessments of genomic instability in cells from each of these categories as a function of numbers of cell divisions post initial radiation exposure.



Using the human hamster hybrid (A_L) cells, which contain a complete set of CHO chromosomes and a single copy of human chromosome 11, the induction of genomic instability among the progeny of bystander cells were examined using the m-BAND assay. Approximately 20% of A_L cells were randomly selected by the image analysis system of the Columbia University microbeam and a precise number of alpha particles were delivered to the nucleus or the cytoplasm of the cells. After irradiation, individual cells were cloned and expanded in cultures. The presence of chromosomal re-arrangement on the human chromosome 11, which include translocation, duplication, paracentric or pericentric inversion, insertion, interstitial or terminal deletions together with whole chromosome 11 deletion or duplication were determined. There is evidence that the fraction of induced abnormal cells involving human chromosome 11 increases at 10 days post-irradiation (~15 population doublings) in the progeny of bystander cells (14.5%), in progeny of nuclear-irradiated cells (14.5%) and in progeny of cytoplasmic-targeted cells (9.7%) relative to the corresponding controls at 6.8%. These data indicate that genomic instability manifest following ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

3. Earlier studies under the auspices of this Program Project [see Project 1] provided evidence of long range biological effects [apoptosis and micronuclei] in bystander cells in 3-D human tissues. Ongoing studies are examining the molecular components of the contributory pathways to this bystander response, and the approach adopted and supportive results are shown in the above Figures. It is readily apparent that the response of individual bystander cells over control levels is enhanced up to a millimeter or more from the region of microbeam delivered alpha particles. Assessments of phosphorylation profiles in this manner will allow for the determination of downstream signaling steps involving the arachadonic acid cascade and/or other pathways controlling the radiation-induced bystander effect. This endeavor will complement the approaches taken in Project 1 and project 2 in fulfillment of the aims of this Program Project.