Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons

Subpopulations of mouse lens epithelial cells, differing in proliferative status, were irradiated with either X rays or fission spectrum neutrons given singly or in four weekly fractions. After various times, epithelia were mitogenically stimulated by wounding and DNA synthesis responses were determined by incorporation of [3H]thymidine. At 1 h following both X and neutron irradiations, significant suppression of the wound response after single doses and a sparing effect of fractionation were evident in both the mitotically quiescent and the slowly proliferating subpopulations. At 1 week following single or fractionated doses of both radiations, recovery was evident in both subpopulations. By 4 weeks, the quiescent subpopulation showed significant recovery after both single and fractionated doses of X rays or neutrons. In contrast, a marked decreased ability to respond after neutron irradiation and, in addition, a significant enhancement effect of neutron fractionation were observed for the slowly proliferating subpopulation. Per gray, neutrons were about 7.5 times more effective than X rays as a single dose and 25 times more effective in four equal fractions. The shift from an initial sparing to a final enhancing effect of neutron fractionation for the slowly proliferating subpopulation has importance for understanding divergent early and late radiation responses following dose fractionation.     

Influence of ionizing radiation on oxygen profiles in different types of multicellular spheroids

Human glioma (U-118 MG and U-138 MG), human colorectal adenocarcinoma (HT-29), human thyroid carcinoma (HTh 7), and hamster embryonic lung (V79-379A) spheroids were irradiated with either single doses of 16 or 40 Gy or fractionated doses of eight times 5 Gy. Oxygen profiles in the spheroids were measured with microelectrodes at different times following irradiation, and these profiles were then compared with the oxygen profiles measured in parallel cultured nonirradiated spheroids. No significant radiation-induced changes in the oxygen profiles were seen in any of the spheroids within the first few days after irradiation. The glioma spheroids did not show any significant increase in oxygen tension even after longer times; however, they were growth inhibited, and the number of S-phase cells was strongly suppressed. Increases in oxygen tension did occur in the HT-29 and V79-379A spheroids but only appeared more than a week after irradiation, when degeneration had started. Histological changes and decrease in diameter were seen in the spheroids that started to degenerate about 5 days after irradiation. Thus radiation doses in the therapeutic range did not, for the spheroids studied, produce rapid increases in the oxygen tension. When a change occurred, it appeared rather late and was probably a consequence of cell degeneration.     

Repopulation kinetics of rat rhabdomyosarcoma tumors following single and fractionated doses of low-LET and high-LET radiation

Measurements were made of clonogenic cell survival in rat rhabdomyosarcoma tumors as a function of time following in situ irradiation with single or fractionated doses of 225-kVp X rays or with 557-MeV/u neon ions in the distal position of a 4-cm extended-peak ionization region. Single doses of 20 Gy of X rays or 7 Gy of peak neon ions reduced the initial surviving fraction to approximately 0.025 for each modality. Daily fractionated doses (four fractions in 3 days) of either peak neon ions (1.75 Gy per fraction) or X rays (6 Gy per fraction) achieved a cell survival of approximately 0.02-0.03 after the fourth dose of radiation. In the single-dose experiments, significant 5- and 10-fold decreases in the fraction of clonogenic cells were observed between the third and fourth days after irradiation with peak neon ions and X rays, respectively. After the sixth day postirradiation, the residual clonogenic cells exhibited a rapid burst of proliferation leading to doubling times for the surviving cell fractions of approximately 1.5 days. Radiation-induced growth delay was consistent with the cellular repopulation dynamics. In the fractionated-dose experiments with both radiation modalities, a large delayed decrease in cell survival was observed at 1-3 days after completion of the fractionated-dose schedule. Cellular repopulation was consistent with postirradiation tumor volume regression and regrowth for both radiation modalities. The extent of decrease in survival following the four-fraction radiation schedule was approximately two times greater in X-irradiated than in neon-ion-irradiated tumors that produced the same survival level immediately after the fourth dose. Mechanisms underlying the marked reduction in cell survival 3-4 days postirradiation are discussed, including the possible role of a toxic host cell response against the irradiated tumor cells.     

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor induction and life shortening in BC3F1 female mice at low doses of fast neutrons and X rays

Extension of previous investigations at this laboratory regarding life shortening and tumor induction in the mouse has provided more complete dose-response information in the low dose region of X rays and neutrons. A complete observation of survival and late pathology has been carried out on over 2000 BC3F1 female mice irradiated with single doses of 1.5 MeV neutrons (0.5, 1, 2, 4, 8, 16 cGy) and, for comparison, of X rays (4, 8, 16, 32, 64, 128, 256 cGy). Data analysis has shown that a significant life shortening is observable only for individual neutron doses not lower than 8 cGy. Nevertheless, assuming a linear nonthreshold form for the overall dose-effect relationships of both radiation qualities, an RBE value of 12.3 is obtained for the 1.5 MeV neutrons. The induction of solid tumors by neutrons becomes statistically significant at individual doses from 8 cGy and by X rays for doses larger than 1 Gy. Linear dependence on neutron dose appears adequate to interpret the data at low doses. A separate analysis of ovarian tumor induction substantiates the hypothesis of a threshold dose for the X rays, while this is not strictly needed to interpret the neutron data. A trend analysis conducted on the neoplasm incidence confirms the above findings. Death rates have been analyzed, and a general agreement between the shift to earlier times of these curves and tumor induction was found.     

Carcinogenesis in laboratory mice after low doses of ionizing radiation

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D(high-LET) < 5 cGy and D(low-LET) < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.     

Slow clones,reduced clonogenicity,and intraclonal recovery in X-irradiated L5178Y-S cell cultures

Profound, long-lasting growth disturbances and reduced viability and clonogenicity were observed in suspension cultures of L5178Y-S (LY-S) murine leukemic lymphoblasts exposed to 0.25–6 Gy of X rays. In most cases, uncloned cultures grew at a reduced rate for periods corresponding to at least 100 cell generations, even when viability of such cultures returned to the normal level. These disturbances were analyzed in clones isolated using agar-supplemented medium. A slow phenotype was much more frequent among surviving clones isolated from LY-S cell cultures irradiated with 3 Gy of X rays than among clones isolated from nonirradiated controls. Growth of individual LY-S clones was affected to different extents, regardless of the clone's viability. The slowest clones had doubling time twice as long (22 h) as that of the control (10–12 h). More than 100 slow clones isolated from irradiated and nonirradiated cultures were followed for prolonged times, and some of them were further subcloned. The slow clones showed a high degree of heterogeneity, and selection for the slowest clone produced clones with increasing proliferative impairment and decreasing cloning efficiency. These results showed that progeny of X-irradiated LY-S cells contained many slowly growing cells, and that their presence affected the growth rate for scores of cell generations. The prolonged impairment of growth rate, viability, and clonogenicity appeared to depend on heritable lesions that were overcome as a result of intraclonal recovery. All slow clones were capable of such recovery, which for clones derived from irradiated cultures typically required periods corresponding to several scores of, but in some cases > 200, cell generations. Intraclonal recovery was much more rapid in slow clones isolated from nonirradiated cultures. This finding indicated that either slow phenotype depended on different cellular changes in the two groups of clones or mechanisms of intraclonal recovery were affected by radiation.     

Repair capacity and kinetics in spheroids from a lung metastasis of a human soft tissue sarcoma: a growth delay study.

Spheroids grown from the human cell line EF8 of a lung metastasis of a human malignant fibrous histiocytoma were given fractionated irradiation with 60Co gamma rays at passages 31 and 32. The mean diameter of the spheroids at the time of treatment was 250 microns. Growth delay was used as the end point in these studies. Two experiments were carried out to determine the capacity and kinetics of repair of sublethal damage. In the first experiment, one, two, and five fractions were given at three or four dose levels with fixed intervals of 360 min. In the second experiment, schedules with two and four dose fractions and intervals of 0, 20, 60, 120, and 360 min were used, each at two dose levels. Data analysis was performed by a direct method based on the alpha/beta model and first-order repair kinetics of radiation damage. In both experiments, the alpha/beta value of EF8 spheroids was estimated to be about 8 (6-10) Gy. The rate constant of repair, mu, and its 95% confidence interval were estimated to be 0.62 (0.40-0.84) 10(-2) min-1, equivalent to a half-time of repair (T1/2) of 112 (83-172) min. A more detailed analysis of the data of the second experiment revealed a significant dependence of the rate constant of repair, mu, on the total radiation effect induced by the fractionated radiation treatments with short overall times. With increasing level of effect, mu decreased. These data indicate that the half-time of recovery of a human tumor can be longer than that of the surrounding normal tissue, in this case lung, at least for a limited range of doses and for some fractionation schedules.     

Repair but not potentiation observed in mouse lung irradiated with neon ions

The lungs of mice were irradiated with 1, 4, or 7 fractions of X rays or neon ions in a 4-cm spread Bragg peak. Lung function as a function of total radiation dose was tested at 7 and 12 months after irradiation by measuring the resting breathing rate in a whole-body plethysmograph. The isoeffect doses increased sequentially with X rays for 1 through 4 to 7 fractions, demonstrating repair of sublethal radiation injury as previously reported. There was also a significant increase of isoeffect dose with neon ions between 1 and 4 fractions but no further increase at 7 fractions. Thus repair instead of potentiation of radiation injury in lung clearly occurred after neon ion irradiation. The effectiveness of neon ions appeared to be closer to that of neutrons with a mean energy of 8 meV than those with a mean energy of 2.3 meV.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

Dose-rate effects of mammary tumor development in female Sprague-Dawley rats exposed to X and gamma radiation

Mammary tumour development was followed in two experiments involving a total of 2229 female Sprague-Dawley rats exposed to various doses of X or gamma rays at different dose rates. The data for another 462 rats exposed to tritiated water in one of these experiments were also analyzed. The incidence of adenocarcinomas and fibroadenomas at a given time after exposure increased linearly in proportion to total radiation dose for most groups. However, no significant increase in adenocarcinomas was observed with chronic gamma exposures up to 1.1 Gy, and the increase in fibroadenomas observed with chronic gamma exposures at a dose rate of 0.0076 Gy h-1 up to an accumulated dose of 3.3 Gy was small compared to that observed after acute exposures. The incidence of all mammary tumors increased almost linearly with the log of dose rate in the range 0.0076 to 26.3 Gy h-1 for 3 Gy total dose of gamma rays. The effects of X rays appeared to be less influenced by dose rate than were the effects of gamma rays.     

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.     

Heat response of human melanoma multicellular spheroids: comparisons with single cells and xenografted tumors

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.     

Comparison of recovery from potential mitotic abnormality in mitotically quiescent lens cells after X, neutron, and 56Fe irradiations

After exposure to various doses of 250 kVp X radiation, 0.85 Me V fission spectrum neutrons, or 600 MeV/A iron (Fe) particles, mitotically quiescent rat lens cells showed no visible evidence of radiation injury. However, following the mitogenic stimulus of wounding, mitotic abnormalities became evident when responding cells entered mitosis. Latent damage and recovery therefrom were monitored at 3, 7, 14, and 28 days after irradiation. Following doses of 1 to 10 Gy of X radiation, the recovery rate, indicated by a decrease in abnormalities with time, was proportional to dose, and the dose-effect slope decreased exponentially with time. Virtually no recovery occurred during the 28 days after 1.25 to 2.25 Gy of fission neutron radiation. After doses of 0.5 to 3.0 Gy of Fe particles, an increased expression of mitotic damage or recovery than recovery occurred. As a consequence of the differing patterns in time for expression of damage or recovery following X rays and the high-LET radiations, the relative biological effectiveness (RBE) increased from 3.6 to 16 for neutrons and from 2 to 10 for Fe particles over the 28-day observation period.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.     

DNA double-strand break misrejoining after exposure of primary human fibroblasts to CK characteristic X rays, 29 kVp X rays and 60Co gamma rays

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.     

Radiation-induced division delay in 9L spheroid versus monolayer cells

The technique of percentage labeled mitoses was used to compare radiation-induced division delay in 9L rat gliosarcoma cells growing as spheroids or as exponential monolayers. The length of delay induced by each of five X-ray doses was determined as the difference between control and irradiated cultures in the time required to reach the half-height of the first peak of labeled mitoses. Spheroid cells were delayed significantly longer than monolayer cells; the slopes of the dose responses were 32 and 13 min/Gy, respectively. Cells in small spheroids (150 micron diameter) were delayed to the same extent as cells in large spheroids (800 micron diameter). Like the contact effect previously observed as enhanced radiation survival of cells grown as spheroids, the increased radiation-induced delay may be a consequence of the growth of cells in three-dimensional contact.     

Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt2cAMP. Treatment of H35 cells with cAMP or Bt2cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and a depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt2cAMP on radiation-induced cell death was studied during fractionated irradiation with 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt2cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that whereas cAMP had no effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2.