M.N. Cornforth and B.D. Loucas
Dept. Radiation Oncology, University of Texas Medical Branch, Galveston,
TX
The most profound influence that whole chromosome painting has had on radiation cytogenetics is the realization that radiation-induced chromosome exchange aberrations are frequently "complex", meaning they involve the interaction of three (or more) damaged breaks distributed among two (or more) chromosomes. Previously it had been assumed that virtually all such interactions involved the simple pairwise rejoining of breaks. Complex aberrations have been implicated in cancer progression, play a major role in genomic instability, and their unexpectedly high frequency following irradiation has forced a major rethinking about the mechanisms underlying chromosome aberration formation.
Because the study of radiation-induced complex exchanges typically has involved painting only a small number of select chromosomes, ambiguities arise in the resulting staining patterns that confound attempts to estimate the true frequency and true extent of complex rearrangements. For example, many exchanges produce pseudosimple staining patterns which appear to be simple, but are actually complex. While other exchanges can often be identified as being complex by their staining patterns, the number of chromosomes and breakpoints which can be deduced to have participated in the exchange often severely underestimates that which has actually occurred. These ambiguities are largely overcome through the use of mFISH, a combinatorial painting technique that can be used to identify each homologous chromosome pair in the human genome.
One of the most surprising findings from our mFISH studies on both lymphocytes and fibroblasts is that the characteristic curvature in the low LET dose response for chromosome aberrations derives principally (if not solely) from complex aberrations, which is to say that the dose response for simple pairwise exchanges is apparently linear. These results have been viewed as a challenge to the classical cytogenetic doctrine that exchange breakpoints involve the interaction of two (or more) damaged sites through molecular processes employing nonhomologous endjoining; i.e., the interaction is "two-hit in nature". This is because a linear dose response for simple exchanges has been interpreted by some investigators to support the controversial notion that exchanges occur because a single radiation-induced chromosome break enters into an exchange with an undamaged chromosome, a notion perhaps more compatible with a homologous/homeologous mechanisms of recombinational repair.
If such a "one-hit" mechanism is responsible for the formation of simple exchanges, then the linearity in question defines a dose response that, by definition, can be extrapolated to arbitrarily low (e.g., sub-rad) doses with confidence. Such a mechanism also implies that the formation of simple exchanges should be independent of dose rate. In other words, the dose response for simple exchanges should be identical, regardless of the rate at which low LET radiation is delivered. To test this prediction we irradiated noncycling G0 human fibroblasts to 137Cs g-rays under conditions of limiting low dose rate (LLDR). [LLDR is defined as a dose rate for which further reduction in dose rate does not cause additional sparing effects (in this case further reduction in the yields of chromosome aberrations)]. Using mFISH, we found that the acute (high dose-rate) dose response (slope) for true simple exchanges was several fold higher than that obtained under LLDR. Consequently, it is unlikely that the linear shape observed for the acute dose response for simple exchanges derives from a one-hit process. Instead, we argue that the process of complex exchange formation competes for broken chromosome ends that might otherwise become involved in the formation of simple exchanges. Competition for reactive breaks causes warpage in the shape of dose response for simple exchanges which, over a limited range of doses, may give the appearance of linearity with dose. In fact, given that the overall contribution to exchange breakpoints from complex exchanges is almost equal to that from simple exchanges at 4 Gy, competition/warpage must lead to saturation at higher doses, virtually guaranteeing that the observed dose response for simples is anything but linear.
Thus, we conclude that the apparent linearity observed in the acute dose response for simple exchanges does not derive from a one-hit interaction process. On the other hand, such data are not inconsistent with the classical two-hit model. An interesting observation less easily explained by either model relates to the appearance of complex aberrations under LLDR. For both models, the contribution of complexes to overall cytogenetic damage would be expected to fall with decreasing dose and decreasing dose rate; in fact, this does occur. Since aberrations produced under LLDR are assumed to arise exclusively by single track action for both models, it is somewhat surprising that any complex aberrations would be produced at all. Nevertheless, our preliminary estimates are that roughly ten percent of all exchange breakpoints derive from complexes under LLDR, a value that should also be applicable to situations of extremely low doses delivered acutely. [For a possible explanation, see the concurrent poster by R. Sachs, et al., entitled "Computer Simulations of Chromosome Aberration Data for Human Cells Subjected to Low-LET Radiation: Mechanistic Implications".]