Lloyd D. Skarsgard

Low-Dose Hypersensitivity and Increased Radioresistance in a Panel of Human Tumor Cell Lines with Different Radiosensitivity

It is well known that cells of human tumor cell lines display a wide range of sensitivity to radiation, at least a part of which can be attributed to different capacities to process and repair radiation damage correctly. We have examined the response to very low-dose radiation of cells of five human tumor cell lines that display varying sensitivity to radiation, using an improved assay for measurement of radiation survival. This assay improves on the precision of conventional techniques by accurately determining the numbers of cells at risk, and has allowed us to measure radiation survival to doses as low as 0.05 Gy. Because of the statistical limitations in measuring radiation survival at very low doses, extensive averaging of data was used to determine the survival response accurately. Our results show that the four most resistant cell lines exhibit a region of initial low-dose hypersensitivity. This hypersensitivity is followed by an increase in radioresistance over the dose range 0.3 to 0.7 Gy, beyond which the response is typical of that seen in most survival curves. Mathematical modeling of the responses suggests that this phenomenon is not due to a small subpopulation of sensitive cells (e.g. mitotic), but rather is a reflection of the induction of resistance in the whole cell population, or at least a significant proportion of the whole cell population. These results suggest that a dose-dependent alteration in the processing of DNA damage over the initial low-dose region of cell survival may contribute to radioresistance in some cell lines.

Low-dose radiation sensitivity and induced radioresistance to cell killing in HT-29 cells is distinct from the "adaptive response" and cannot be explained by a subpopulation of sensitive cells.

Several reports using two different improved assays of clonogenicity have indicated the presence of a hypersensitive region in the radiation survival response at low doses, followed by an increase in radioresistance, in many mammalian cell lines. Mathematical modeling of these responses has suggested that it is unlikely that this effect can be explained by the presence of a small subpopulation of sensitive cells; however, this possibility cannot be excluded solely on the basis of those results. A second explanation has been offered which hypothesizes that a radiation-induced mechanism causes an increase in cellular radioresistance. This proposal has led to speculation that the substructure observed at low doses in these cell lines is related to the adaptive response, which hypothesizes the induction of a repair mechanism after a small priming dose of radiation which can protect cells against a larger second dose given several hours later. We have investigated these proposals with a study of priming doses using human tumor HT-29 cells, which we have previously shown to exhibit low-dose hyper-radiosensitivity. Our results provide significant evidence that this effect cannot be explained by a subpopulation of sensitive cells. However, the results also suggest that the radiation-induced increase in radioresistance observed in this cell line is distinct from the adaptive response.

The radiation response of asynchronous cells at low dose : evidence of substructure

Flow cytometry and cell sorting techniques have been used together with repeated measurement in an attempt to define better the radiation survival response of asynchronously dividing Chinese hamster V79-171 cells under aerobic and hypoxic conditions. Although the first two decades of cell inactivation have been examined, particular attention has been given to the low-dose range of a few grays, as used in individual radiation therapy treatments. A single linear-quadratic dose-response function was consistently unable to fit both the low-dose and high-dose data satisfactorily, suggesting a two-component response. Separate fitting of the low-dose and high-dose portions of the response yielded alpha and beta values which differed significantly (P = 0.001 to 0.002). The data are consistent with the hypothesis that the observed substructure simply reflects the presence of subpopulations of sensitive (G1-, G2-phase) and resistant (late S-phase) cells, which are resolved in these measurements. These results may have significance for certain situations in radiation therapy and in biophysical modeling of the radiation response.

Ascorbate-enhanced cytotoxicity of misonidazole.

MISONIDAZOLE, an electron-affinic, nitroheterocyclic compound (1-(2-nitro-1-imidazole)-3 methoxy-2-propanol, Ro-07-0582) is undergoing preliminary clinical trials, which are exploring its capacity to sensitise hypoxic tumour cells to ionising radiation damage1–4. The drug also possesses a substantial cytotoxic effect, independent of radiation, which is selectively expressed in hypoxic cells5,6. Both of these properties may contribute to the potential value of this agent as an adjunct to radiation therapy of malignant disease in those situations where radioresistant hypoxic cells represent the limiting factor in achieving local control. It is also possible that the cytotoxic property could be a useful adjunct to chemotherapy as it should be effective against noncycling hypoxic tumour cells. Misonidazole, however, may be cytotoxic to the normal hypoxic tissues in the human body (nerve, skin, cartilage, and so on) and this is a major concern in the clinical application of the drug1,4. Furthermore, misonidazole is metabolised in mammalian cells and at least two products have been isolated7, toxic to both aerobic and hypoxic mammalian cells. Thus, if they are able to diffuse freely, the metabolites may also be a threat to aerobic tissues8. This Cytotoxicity of misonidazole is temperature dependent5,9,10, and varies with cell line11,12, and cysteamine seems to protect against it13. It leads to strand breaks in cellular DNA and those cells which fail to survive also fail to repair these strand breaks12. In this work we show the effect of ascorbic acid (vitamin C) on the cytotoxicity of misonidazole.

The Effect of Misonidazole as a Hypoxic Radiosensitizer at Low Dose

Using an automated low dose survival assay, the radiosensitizing effectiveness of misonidazole at low radiation dose (0-6 Gy) was measured in cultured mammalian cells. Also measured was its effectiveness at high doses of radiation (0-35 Gy) using the conventional survival assay. In both cases, several concentrations of the drug from 0 to 5 mM were used. The data at low doses were analyzed by a two-parameter mathematical equation with linear and quadratic dose terms, S = e-alpha D-beta D2, which proved to be a good fit to the experimental data at all misonidazole concentrations. It is shown that whereas the coefficient of the quadratic dose term, beta, increases significantly with increasing misonidazole concentration, the drug does not significantly affect the coefficient of the linear term, alpha. The enhancement ratio (ER) of misonidazole is shown to be decreased at lower doses. The clinical implications of this result are discussed.

Reduction of misonidazole and its derivatives by xanthine oxidase

The nitroimidazole drug misonidazole, now undergoing clinical trials as a radiosensitizer of hypoxic cells, is selectively toxic to hypoxic mammalian cells; this toxicity may be due to metabolic reduction of the drug. Zinc reduction of misonidazole yields its azo and azoxy derivatives [P. D. Josephy, B. Palcic and L. D. Skarsgard, in Radiation Sensitizers (Ed. L. W. Brady), p. 61. Masson, New York (1980)]. We have shown in the present work that misonidazole and its azo and azoxy derivatives were reduced by xanthine oxidase, under hypoxic conditions. The nature of the products of misonidazole reduction was examined; hydroxylamino-misonidazole appeared to be the main product.

Radiosensitization of Mammalian Cells by Misonidazole and Oxygen: DNA Damage Exposed by Micrococcus luteus Enzymes

When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37/sup 0/C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract.

Radiosensitization of hypoxic cells at low doses.

We have measured the radiosensitization produced in hypoxic V79 cells in vitro by SR 2508, Ro-03-8799, and oxygen at conventional doses (0-30 Gy) as well as at low doses (0-4 Gy). Two techniques have been used: (a) FACS (Fluorescence Activated Cell Sorter) plating followed by the conventional colony formation assay and (b) microscopic scoring of surviving and non-surviving cells using the DMIPS (Dynamic Microscope Image Processing Scanner) system. The results show that the enhancement ratios (ER) for both drugs and for oxygen are dose dependent, decreasing somewhat at doses less than 4 Gy. The low dose sensitization measured at S = 0.8 for SR 2508, Ro-03-8799 and O2 was found to be approximately 73%, 61% and 75% of the high dose sensitization for these three agents, respectively. These values are not significantly different. Both the alpha- and beta-components of cell kill appeared to be sensitized by all three agents. The enhancement ratios obtained by the two different techniques are in general agreement, though some qualitative differences have been observed.

Substructure in the radiation survival response at low dose in cells of human tumor cell lines.

In earlier studies using asynchronously growing Chinese hamster cells, we observed substructure in the survival response at low doses. The substructure appeared to result from subpopulations of cells having different, cell cycle phase-dependent radiosensitivity. We have now applied the same flow cytometry and cell sorting technique to accurately measure the responses of cells of eight different asynchronously growing human tumor cell lines, representing a wide range in radiosensitivity. When the data were fitted with a linear-quadratic (LQ) function, most of these lines showed substructure similar to that observed in Chinese hamster cells, with the result that values of alpha and beta were dependent on the dose range used for fitting. Values of alpha describing the low-dose response were typically smaller (by as much as 2.2 times) than the alpha describing the high-dose response, while values of beta were larger at low doses. Values of alpha/beta from our measurements are in reasonable agreement with other values published recently if we fit the data for the high-dose range (excluding, for example, 0-4 Gy), which corresponds to a conventional survival response measurement. However, the values of alpha/beta describing the low-dose range were, on average, 2.8-fold smaller. The results show that the usual laboratory measurement of cell survival over 2 or 3 logs of cell killing, if fitted with a single LQ function, will yield alpha and beta values which may give a rather poor description of cell inactivation at low dose in asynchronous cells, no matter how carefully those measurements are done, unless the low-dose range is fitted separately. The contribution of killing represented by the beta coefficient at low doses was found to be surprisingly large, accounting for 40-70% of cell inactivation at 2 Gy in these cell lines. A two-population LQ model provides excellent fits to the data for most of the cell lines though, as one might expect with a five-parameter model, the best-fitting value of the various parameters is far from unique, and the values are probably not reliable indicators of the size and radiosensitivity of the different cell subpopulations. At very low dose, below 0.5-1 Gy, another order of substructure is observed: the hypersensitive response; this is described in the accompanying paper (Wouters et al., Radiat. Res. 146, 399-413, 1996).

Pions and pig skin: Preclinical evaluation of RBE for early and late damage

The skin of 50 pigs has been irradiated with negative pi mesons and with X rays in order to determine the RBE for early epidermal and later dermal damage. Late fibrosis was not studied. Four, 7, 9 and 10 fractions were used. An estimate of the RBE was made from the reactions on each pig for both early and late damage so that interanimal variability would be avoided. The data were also averaged to obtain mean dose response curves. There was no tendency for higher RBEs for late than for early skin damage. These pig studies have demonstrated an RBE of about 1.5 for early epidermal reactions and a slightly lower RBE (approximately 1.4) for later dermal damage in the same animals. This indicates that at doses of about 2.0 to 3.5 Gy pions, the medium wave skin damage is unlikely to be more severe than would be predicted from the early skin reactions and the accumulated clinical experience with X rays. However, if the trend to a steeper slope for the RBE versus dose per fraction for late injury is correct, as indicated by other published studies a relative increase in the late injury might be expected if much lower doses per fraction are used. The present clinical studies at Vancouver using 15 X 2.1 Gy pions indicate that an RBE of 1.5 is appropriate for epithelia, brain and colorectum.