Tor Brustad

X-ray inactivation of human cells in tissue culture under aerobic and extremely hypoxic conditions in the presence and absence of TMPN.

SummaryA technique is described which permits irradiation of suspensions of human cells in tissue culture either aerobically (equilibrated with air) or under extremely hypoxic conditions (flushed with a mixture of 97 per cent N2 and 3 per cent CO2, the oxygen content of which was less than 15 p.p.m.).An established cell-line, NHIK 3025, originally derived from an early stage of human cancer of the cervix, was used. The D0 value of these cells under aerobic conditions was 130 rads and the extrapolation number about 3. Under extremely hypoxic conditions, the dose-effect curve was exponential with a D0 value of 600 rads. The o.e.r. derived from the ratio of the D0 values was therefore 4·6.2,2,6,6tetramethyl-4-piperidinol-N-oxyl (TMPN) at a concentration of 10−2 M exerted a pronounced sensitizing effect under extremely hypoxic conditions (TMPN e.r. = 2·5), and yielded dose-effect curves with an extrapolation number of 3. The high radio-resistance of the cell under extremely hypoxic conditions in the absence o...

Radiation response of multicellular spheroids initiated from five human melanoma xenograft lines. Relationship to the radioresponsiveness in vivo

Multicellular spheroids, initiated from five human melanoma xenograft lines (E.E., E.F., G.E., M.F., V.N.) and grown in liquid-overlay culture, were characterised with regard to radiation response. The principal aim of the work was to search for possible correlations with the radioresponsiveness in vivo of the parent xenografts. The spheroids were 100 +/- 5 microns in diameter at irradiation and did not contain radiobiologically hypoxic cells. Single-cell survival measured in soft agar, specific growth delay and spheroid cure were used as end-points. The cellular radiosensitivity was the same whether a melanoma was grown as spheroids or as xenografts. An intercellular contact effect was found for spheroids of the G.E. melanoma but not for spheroids of the E.E., E.F., M.F. and V.N. melanomas, in agreement with previous observations from studies of the corresponding xenografts in vivo. A positive correlation was found between the radiation response of the spheroids, measured as cell survival after 6 Gy or as specific growth delay after 6 Gy, and the radiation response of the parent tumours, measured as specific growth delay after 15 Gy. There was no correlation between the SCD50 (the dose required to cure 50% of the spheroids) and the radioresponsiveness in vivo. It is concluded that differences in radioresponsiveness in vivo among tumours may be identified from studies of the corresponding multicellular spheroids grown in liquid-overlay culture.

REACTIONS BETWEEN ORGANIC NITROXYL FREE RADICALS AND RADIATION-INDUCED TRANSIENTS IN THE DNA BASES.

Second-order rate constants (k′) have been determined by pulse-radiolysis technique for reactions of OH-induced transients in each of the four DNA bases with three organic nitroxyl compounds, thus: The structural formula of the nitroxyl compounds, which are known to enhance specifically the radiation-induced lethality of anoxic cells, are as follows: The reactions have been studied in 2 x 10-3 M phosphate buffer, pH 7 and the results are presented.

Survival curves after X-ray and heat treatments for melanoma cells derived directly from surgical specimens of tumours in man

X-ray and heat survival curves were established for melanoma cells derived directly from surgical specimens of tumours in man by using the Courtenay soft agar colony assay. The plating efficiency for 11 of the 14 melanomas studied was sufficiently high (PE = 0.3-58%) to measure cell survival over at least two decades. Experiments repeated with cells stored in liquid nitrogen showed that the survival assay gave highly reproducible results. The melanomas exhibited individual and characteristic survival curves whether exposed to radiation or heat (43.5 degrees C). The Do-values were in the ranges 0.63-1.66 Gy (X-rays) and 33-58 min (heat). The survival curves were similar to those reported previously for human melanoma xenografts. The radiation sensitivity of the cells was not correlated to the heat sensitivity. Since the melanomas appeared to be very heterogeneous in radiation response in vitro as melanomas are known to be clinically, it is suggested that melanomas may be suitable for prospective studies aimed at establishing whether clinical radioresponsiveness somehow is related to in vitro survival curve parameters.

On the binding of an organic nitroxide free radical to radiation-induced desoxyribonuclei acid (DNA) radicals under anoxic conditions

Abstract Rapid mixing techniques have been used to study radiation-induced complex formation between desoxyribonucleic acid (DNA) and tritiated 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) under anoxic conditions. Radiation-induced 3H-TAN-DNA complexes were separated from TAN and low molecular weight TAN products on a Sephadex G-25 column and the extent of binding of TAN to DNA determined from the 3H-activity profiles. No complex formation is observed when TAN is irradiated and at various times, Δt, afterwards (Δt>10 ms) mixed with unirradiated DNA. Considerable complex formation is observed when DNA is irradiated and at various times afterwards mixed with unirradiated TAN. The data prove unequivocally that complex formation results from reactions between TAN and radiation-induced DNA transients. Assuming that the decay of the DNA transients is a first-order process, the data indicate that two DNA transients with different half-lives ( T 1 2 ′ ≈ 10 s and T 1 2 ″ ≈ 70 ms ) are involved.

Reactions between Nitroxyls and Radiation-induced Long-lived DNA-transients

SummaryThe reactions of the organic nitroxyl free radical 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) with radiation-induced DNA transients have been studied under anoxic conditions by means of a special irradiation-mixing technique.Unirradiated 3H-TAN (2 × 10−5 M in 10−2 M NaCl) was mixed in proportion 1 : 1 with irradiated DNA (1 mg/ml in 10−2 M NaCl) at predetermined times (t) after irradiation, with t > 20 sec. After irradiation and mixing, the per cent loss of TAN spins (NSL) was determined by E.S.R. technique, and the per cent 3H-TAN bound to DNA (NB) was determined by column chromatography.The data show that TAN interacts with radiation-induced DNA transients in two different ways,: where DNA· are free radicals and the (DNA−TAN)complexes are void of TAN spins, and The latter reaction involves electron transfer from radiation-induced DNA transients (DNAtr) to TAN, yielding reduced TAN and DNA derivative (DNA+1).The data show that NSL/NB ≈ 2·5 when 20 sec < t < 40 min. About the same ratio was foun...

Binding of Nitroxyls to Radiation-induced DNA-transients in E. Coli K–12 under in Vivo Conditions and Its Relevance for Radiosensitization

SummaryBinding of tritiated 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (3H-TAN) to radiation-induced DNA-transients in E. coli K–12 strain AB 2463 rec−A, has been studied under in vivo conditions. In N2O-saturated cell suspensions 30 per cent higher yield is observed than in N2. Aerobic bacteria show slight binding ability.The time-course of the ability of TAN to (a) form complexes with DNA and (b) act as an anoxic cell radiosensitizer has been investigated when TAN is added to the cell suspension at different times before and after irradiation.When TAN is added before exposure, the yield of TAN-DNA complexing is reduced by only 40 per cent when the contact time is reduced from 10 min to 200 msec. For contact times as short as 200 msec, however, only a minute radiosensitization is observed.When TAN is added after exposure, there are significant binding yields when mixing occurs within 2 sec after irradiation of the cell suspension. No sensitization at all results, however, even when TAN is added only 200 mse...

Radiosensitization of anoxic cells of E. coli K-12 by organic nitroxyl free radicals.

SummaryRadiosensitization of anoxic, log-phase cells of E. coli K-12, strain AB 2463 rec−A, by organic nitroxyl free radicals has been studied by means of two kinds of rapid-mixing equipment.For the sensitizer concentrations used (10−3 M) the nitroxyl-mediated radiosensitization is shown to be nearly independent of the dose-rate from 20 rads/sec to 8 × 109 rads/sec. Insignificant sensitization was observed when cells were irradiated, and at various times t afterwards (where t > 10 m sec), mixed with an unirradiated solution of nitroxyls.The time course of the nitroxyl-mediated radiosensitization resulting from irradiation performed at given times after mixing of the cell suspension with the nitroxyl solution was determined. It suggests that three processes are involved:(1) A very rapid one, perhaps associated with membrane transients, which for 10−3 sec < t < 10−1 sec provides a small sensitization (about 30 per cent increase).(2) An intermediate process, perhaps governed by diffusion of nitroxyls into th...

Reactions between nitroxyl free radicals and radiation-induced transients in nucleosides.

SummarySecond-order rate constants have been determined by pulse radiolysis for reactions of the three organic nitroxyl compounds TAN, TMPN and NPPN with OH-induced transients in four nucleosides at pH 7. The results are presented and compared with the rate constants for the reactions with the corresponding DNA-bases.

Differences in thermosensitization among cloned cell lines isolated from a single human melanoma xenograft

Differences in thermosensitization (effect of step-down heating) among one uncloned and five cloned cell lines isolated from a single tumor of a human melanoma xenograft were studied. Cells from passages 7-12 in vitro were exposed to graded heat treatments at 41.5 degrees C immediately, 1 h, and 2 h after a conditioning treatment of 43.5 degrees C (90 min). The thermosensitization was largest immediately after the conditioning treatment and then decayed exponentially. The differences among the cell lines were reflected in the maximum magnitude as well as in the rate of decay of the thermosensitization. The maximum thermosensitization ratios (TSR), calculated as the ratio of the D0 values at 41.5 degrees C for single-heated and preheated cells, ranged from 5.3 +/- 1.5 to 14.9 +/- 5.2 and were not correlated to the surviving fractions after the conditioning treatment. The half-times for the decay of the thermosensitization ranged from 1.5 +/- 0.3 h to 3.1 +/- 0.5 h and were not correlated to the maximum TSR. Moreover, there was no correlation between the magnitude of the maximum thermosensitization at 41.5 degrees C and the magnitude of the maximum thermotolerance at 43.5 degrees C, as induced by the same treatment (43.5 degrees C for 90 min).