Charles R. Geard, Gloria Jenkins-Baker,
Brian Ponnaiya., Mutian Zhang, Alan Bigelow and Gerhard Randers-Pehrson
Radiological Research Accelerator Facility, Center for Radiological Research,
College of Physicians and Surgeons of Columbia University, lrvington.
New York, USA, 10533
The selective exposure of parts of intact individual cells to high-energy radiation was recognized many years ago by Zirkle and Bloom [1] as having the potential to provide information about the mechanisms whereby radiation produces such "strikingly injurious effects in living systems", and to "aid in analyzing the normal functions of the various cell parts by selectively altering them.". Accelerator based microbeams and collimated or micro-needle coated isotopic alpha particle sources helped to establish one of the basic paradigms of radiation biology; that it is the hit cell nucleus from which deleterious effects originate. In 1962 a Symposium on partial and microbeam irradiation reported on by Smith [2], was held at which, "All participants were persons who are actively interested or working in this rapidly growing field." It now appears that the growth of the field stalled for a number of years, with a resurgence in interest developing in part from concern about the effects of single alpha particles and from low dose studies with isotopic alpha particle sources. The results of these latter studies challenged the basic paradigm of radiation action. Low fluences of alpha particles were delivered to mammalian cells such that only a small fraction of cells were likely to have had their nuclei impacted by an alpha particle. Significantly more cells showed changes in sister chromatid exchange frequencies, in chromosomal changes and in gene expression than were traversed by alpha particles, hence some responding cells were bystanders of hit cells. These endpoints
along with other cellular/molecular endpoints with relevance to the risks associated with exposure to ionizing radiation were not available in the early sixties. The development of pertinent risk and mechanistically oriented assays may perhaps be considered to be the greatest incentive for the burgeoning current interest in microbeams.
Studies with broad beam alpha particle sources, either isotopic or accelerator based, can not of course readily distinguish between known hit cells and known bystander cells, nor can the relative contributions of nuclear hits, of cytoplasmic hits or even of media hits to the final biological response be ascertained. The ability to establish the origin of individual cell biological responses is however attainable with a microbeam, consistent with the original expectations of Zirkle and Bloom [1].
We have used the Columbia University RARAF microbeam and specifically devised protocols to target individual cell nuclei, or cell cytoplasm, and to miss cells completely hitting intercellular medium. Using these protocols all sites or a known proportion of sites can be irradiated with a precisely known number of alpha particles. That is, some cells can be hit cells while the remaining proportion will be bystander cells.
Alternatively, cells can be labeled with 2 vital dyes then plated in known proportions and only one cell population irradiated. Following irradiation the hit cells can be discriminated from the bystander cells, with the relative positions of each cell at the time of irradiation being recorded. Endpoints examined include frequencies of micronuclei, cell growth by monitoring cell numbers [all cells on a dish are examined], progression of cells through the cell cycle by monitoring bromodeoxyuridine uptake, and the expression of the stress related genes, p53 and p21[WAFI/Cipl]. The latter is undertaken on cells in situ using immunofluorescence, and also following the removal of individual cells of known radiation history with a micromanipulator and carrying out single cell RT-PCR.
The conclusions from an extensive series of studies with normal human fibroblasts using RARAF microbeam protocols are as follows:
1. Deliberately missing cells and irradiating the culture medium between them produces no detectable response.
2. Irradiating cell cytoplasm [including the medium surrounding the cell] produces no detectable increase in the frequency of micronuclei, but does induce some cell cycle delay in synchronized GO/GI phase cells. No delay is seen in cycling cells.
3. Irradiating cell nuclei [including surrounding cell cytoplasm and medium] results in fluence dependent increases in micronuclei, cell cycle delay and increases in gene expression. This response specifically includes exactly I alpha particle.
4. Irradiating 50% of cells through their nuclei produces a response in known non-hit bystander cells which is not dependent on the number of particles through hit cells.
5. Reducing the proportion of hit cell nuclei results in a proportional lessening of response in bystander cells.
6. Irradiating hit cells through cytoplasm does not produce a response in bystander cells.
The expression of a bystander effect in non-hit cells originates from insult to the nuclei of hit cells.
References:
1. Zirkle, RE, and Bloom, W. Irradiation of parts of individual cells. Science 117: 487-493, 1953.
2. Smith, CL. Effects of partial and microbeam irradiation of cells. Nature 196: 728-729, 1962.