Mortimer M. Elkind

DNA damage and cell killing. Cause and effect

The evidence supporting a cause and effect relationship between DNA damage and cell killing is examined in the light of what is currently known about the organization and replication of genomic DNA in eukaryotic cells and the radioenergetics of DNA breakage. A large disparity is identified between characteristic doses for cell killing and for the production of DNA lesions (i.e., single‐ or double‐strand breaks). In contrast, the sensitive phase of the inhibition of DNA synthesis has a dependence on dose quantitatively similar to that of cell killing. A model is developed in which single‐ and double‐strand breaks are associated with the inhibition of replicon initiation, whereas only double‐strand breaks are primarily responsible for strand elongation. Furthermore, the model points to the replisome and the region of replicated DNA just downstream from the fork as the locus of radiation action.

The “recall effect” in radiotherapy: Is subeffective, reparable damage involved?

It has been proposed that lethal mutations among the progeny of a surviving cell could be the basis for the recall effect when chemotherapy is applied subsequent to the repair of normal-tissue injury resulting from a course of radiation therapy. Because radiotherapy is usually multifractionated, the possibility exists that repair of heritable injury of this type could occur between fractions as is the case for sublethal damage. To examine this possibility, the endpoint small-colony formation was used--an endpoint which integrates the effects of a number of radiation-induced aberrancies including lethal mutations--and low-dose-rate irradiation. It was found that, even after net surviving fractions comparable to those sought in radiotherapy were reached, little damage remained expressible as a deficiency in the size of the colony generated from a surviving cell. We conclude that the damage expressible as a lethal mutation is reparable and therefore the recall effect must be attributed to some other cellular mechanism.

Cell-cycle sensitivity, recovery from radiation damage and a new paradigm for risk assessment

Tikvah Alpers interest in science was broad, from scrapie to mammalian cells and cancer. Much of her own work focused on cell lethality, like that of many other radiobiologists, but this was natural because of the simplicity of the endpoint cell survival and its relevance to cancer therapy. Tikvah had broader interests, however, that included the effects of radiation on living systems in general like the induction of cancer and the cellular and molecular processes contributing to it. In this essay, some ideas are developed that lie in the mainstream of her interests. Starting with functional measures of the recovery or repair from radiation damage, a role for repair is illustrated in connection with mutagenesis and neoplastic transformation both discussed in the context of radiation-induced cancer. These topics are central to a model explaining the anomalous enhanced neoplastic transformation and cancer observed when low doses of a high-LET radiation are protracted in time. Under particular circumstances, the formalism of the model predicts application to protracted low-LET exposures as in the instance of repair-deficient target cells and sporadic breast cancer. The latter discussion leads to the proposal that the paradigm in current use for evaluating cancer risk should be broadened: from a simple dose-effect relation to one that includes cell kinetics (during protracted exposures), cell-cycle dependencies, and the influence of cellular repair or the lack thereof.

Novobiocin inhibits the repair of potentially lethal damage but not the repair of sublethal damage.

Because of the critical role of the DNA topoisomerases in the synthesis and conformation of DNA, and the well-known observation that radiation inhibits replicative DNA synthesis, we have examined the possibility that inhibitors of these enzymes might influence radiation lethality. In particular, using protocols involving the administration of either fresh or conditioned medium, we examined the ability of intercalative and nonintercalative inhibitors to affect the expression of potentially lethal damage and/or sublethal damage. The inhibitors examined were amsacrine, teniposide, etoposide, and novobiocin; only the latter compound was clearly effective in a selective way at nontoxic concentrations, and this was observed specifically in reference to the repair of potentially lethal damage effected by incubation in conditioned medium. These results are another example of differences between the repair of sublethal versus potentially lethal damage that further support distinctions between the two. At a mechanistic level, these and other data suggest that the property of novobiocin that is relevant in the foregoing is its metabolic inhibition of replicative DNA synthesis, a process which may be more important in the repair of potentially lethal damage as opposed to sublethal damage.

Neoplastic Transformation of C3H Mouse Embryo Cells, 10T1/2: Cell-cycle Dependence for 50 kV X-rays and UV-B Light

The variation of neoplastic transformation induced by 50 kV X-rays, and by solar-simulating UV-B light, was studied through the cell cycle of C3H mouse embryo cells designated 10T1/2. A mitotic shake-off method was used to harvest mitotic cells. The progression through the cell cycle of initially mitotic cells was followed as a function of time by flow cytometry, DNA labelling for passage through S-phase, and growth curves for cell number. At 2-3 h after shake-off, about 90% of the cells were in early G1-phase and by 15 h 60-70% of cells had reached S-phase. For 2.5 Gy, the transformation frequency per viable cell in M-phase was some five times higher than in S-phase. In contrast, at similar survival levels, UV-B light is less efficient in transforming mitotic cells. For both types of radiation, the frequency of neoplastic transformation per viable cell was roughly inversely proportional to survival.

Radon-induced Cancer: A Cell-based Model of Tumorigenesis Due to Protracted Exposures

In 1982, results with C3H mouse embryo cells showed that the frequency of neoplastic transformation was enhanced when exposures to fission-spectrum neutrons were protracted in time. This finding was unexpected because the opposite was found with low-LET radiations. Similar neutron enhancements were reported with normal life-span Syrian hamster embryo cells, and with human hybrid cells. Because other studies did not confirm the preceding, in 1990--at a conference convened by the US Armed Forces Radiobiological Research Institute--a biophysical model was proposed to explain the basis for the enhancement observed in some experiments but not in others. The model attributed special sensitivities, related to killing and neoplastic transformation, to cells in and around mitosis. Subsequently, it was shown that late G2/M phase cells constituted this window of sensitivity. In the instance of tumorigenesis, the model predicted that protracted exposures to a high-LET radiation would result in enhanced frequencies of transformation providing that susceptible cells were cycling or could be induced to cycle. The model explained data on lung tumour induction in rats breathing radon at different concentrations, and uranium miners working in atmospheres containing different concentrations of radon. The model also explains the anomalous finding that lung cancer deaths are often sublinearly correlated with indoor radon concentration.

Inhibitors of poly (ADP-ribose) synthesis inhibit the two types of repair of potentially lethal damage

PURPOSEnThe purpose of this study was to examine whether 3-amino-benzamide (3ABA), an inhibitor of poly (ADP-ribose) synthesis, inhibits the two types of potentially lethal damage (PLD) repair, termed slow and fast.nnnMETHODS AND MATERIALSnThe fast-type PLD repair was measured by the decrease in survival of V79 Chinese hamster cells by postirradiation treatment with 3ABA. The slow-type PLD repair was measured by the increase in survival by posttreatment with conditioned medium (CM), which became conditioned by growing a crowed culture of cells and supports the slow-type PLD repair.nnnRESULTSnUp to 1 mM, 3-ABA inhibited the slow type repair; at doses of 2 mM and above, it inhibited the fast type of PLD repair.nnnCONCLUSIONnThere are quantitative differences in cellular effects of 3ABA dependent on concentration. Poly (ADP-ribose) appears to play an important role in the PLD repairs and has little effect on the repair of sublethal damages.

Additivity of cytotoxic damage due to dimethylbenz(a)anthracene and X rays.

7,12-Dimethylbenz(a)anthracene is one of a group of polycyclic aromatic hydrocarbons that are known to be indirectly acting carcinogens. As a product of the incomplete combustion of complex hydrocarbons, dimethylbenzanthracene is present in the environment and may therefore act on living systems in conjunction with ionizing radiation. We have studied the cytotoxic effects of dimethylbenzanthracene by itself, together with other polycyclic aromatic hydrocarbons, and combined with X radiation. Pre- or postirradiation treatment of mouse C3H 10T1/2 cells with dimethylbenzanthracene progressively removes the shoulder of the X-ray survival curve and, consistent with that observation, the survival sparing from dose fractionation is progressively lost. The cotreatment of cells with 3-methylcholanthrene and dimethylbenzanthracene largely abrogates the killing due to the latter compound alone and, accordingly, returns the shoulder to the survival curve. The application of dimethylbenzanthracene, or similar compounds, between X-ray dose fractions, separated by 4 h, is without effect quite likely because of the need for metabolic activation of the compound for effectiveness. Dimethylbenzanthracene is believed to be genotoxic because, after it is activated, it forms bulky adducts with DNA. Hence these results suggest that bulky adducts are a form of DNA damage operationally equivalent to sublethal X-ray damage.

Radiobiology in radiotherapy: A personal history

It all started for me when I was offered the opportunity to do graduate work at the Massachusetts Institute of Technology. At that time, I was a mechanical engineer under Leo Marinelli in the Physics Department of the Sloan-Kettering Institute/Memorial Hospital. In 1949, Egan Lorenz, a biophysicist at the National Cancer Institute, offered to have me added to the staff of that Institute if I was willing to join John Trump’s group at MIT to learn how to service Van de Graaff accelerators. I agreed and during the next four years I did, in fact, learn about the care of a type of particle accelerator that was then becoming a useful machine for supervoltage therapy. However, I also did a Master’s and a Doctorate, the latter in physics. As a result .when, in 1953, Lorenz broached the possibility of my also learning some radiobiology, I seized the opportunity and, still with support from NCI, I went off to Berkeley to join Cornelius Tobias at the Donner Laboratory of the University of California. Tobias headed a group which at that time was pursuing fundamental studies with yeast cells. My move to Berkeley was a debut, since having had no formal training in biology, I was indeed an innocent looking for an identity. During the succeeding nine months. Lorenz succombed to a heart attack and I was called to Bethesda to start servicing high-voltage machines. Although I was prepared to fulfill my part of the understanding that I had with Lorenz, I did not relish this assignment since the brief taste of biological research that I had had whetted my appetite. Fortunately for me, G. Burroughs Mider was the Scientific Director of NCI. When he learned that what I really wanted to study was radiobiology. he offered the encouragement and. support that enabled me to obtain an assistant, Harriet Sutton, and to set up my own laboratory. In brief. therefore, that is how 1 made the transition from mechanical engineering, Mortimer M. Elkind. Ph.D.

Transformation-sensitive Cells in G2/M-phase are Not Promoted by TPA Following 137Cs γ-rays

Mouse C3H 10T1/2 cells are most sensitive to radiation-induced neoplastic transformation in the G2/M-phase of the cell cycle. When synchronized 10T1/2 cells were exposed to phorbol 12-myristate 13-acetate (TPA) after irradiation, transformation of cells not in the transformation-sensitive window was enhanced, but transformation of cells already in the transformation-sensitive window was not. Earlier work showed that (a) TPA enhances the frequency of transformation of both high and low dose-rate γ-irradiated cells by about the same factor, but that (b) TPA enhances the transformation of cells exposed to low dose-rate fission spectrum neutrons appreciably less than cells exposed to high dose-rate fission spectrum neutrons. The latter observation is consistent with the inability of TPA to promote cells in the transformation-sensitive window and with the role of such cells in enhancing transformation by protracted doses of neutrons. The data provide a cellular basis for studying the biochemical/molecular aspe...