Cultured TN-368 lepidopteran insect cells exhibit a pronounced resistance to the lethal effects of a variety of physical agents, including X rays and 254 nm UV light, as well as a large number of chemicals. The resistance to ionizing radiation has previously been associated with an inducible process which is not expressed in unirradiated cells or cells receiving less than some minimal amount of radiation necessary for activating the process. The studies in this paper were initiated in an attempt to identify and characterize the inducible proteins associated with the marked radiation resistance of the TN-368 cells. Cells were exposed to doses of 0, 25, 64 or 350 Gy of 137 Cs γ rays and incubated either for 3 h in medium containing [ 35 S]methionine or for 2 h without labeling. Labeled cells were separated into nuclear and cytoplasmic fractions and proteins were analyzed on two-dimensional polyacrylamide gels. Unlabeled cells were used to isolate total RNA which was translated in vitro in a rabbit reticulocyte lysate system with 35 S label. These translation products were also analyzed by two-dimensional electrophoresis. Gamma irradiation of the TN-368 cells resulted in the de novo synthesis of several proteins as well as the complete inhibition of others. The number of such proteins identified was 19. These proteins ranged in size from 18-73 kDa, with a pI distribution of 4.7 to 6.1. In addition to the unique proteins, a large number of other proteins were also either up- or down-regulated. These observations were made in both nuclear and cytoplasmic fractions as well as in the translation products of RNA produced after irradiation. These studies indicate that RNA and protein synthesis in lepidopteran cells are coordinately regulated in response to ionizing radiation and may participate in the pronounced radioresistance of the TN-368 cells.

Two γ-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the γ-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137 Cs γ rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P < 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P < 0.0001 and P < 0.003 levels, respectively. In cross-sensitivity testing of the γ-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44°C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44°C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.

TN-3681 lepidopteran insect cells display a pronounced resistance to the lethal effects of ionizing radiation and exhibit superior DNA repair capabilities. When a TN-368 cell population entering stationary growth phase is irradiated with 137 Cs γ rays and then incubated for several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation. Similarly, the survival of cells plated immediately following the second of two equivalent doses separated by several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses. Both processes exhibit similar biphasic repair kinetics and reach maximal levels by 6 h. The phenomena appear initially to be analogous to confluent-holding and split-dose recovery as described for mammalian cells. However, the survival levels obtained for doses of 61-306 Gy after allowing for these recovery processes to occur are quite high and greatly exceed survival levels for all but relatively low doses less than 50 Gy. For example, while the survival of cells irradiated with 150 Gy is near 0.15, the survival of cells receiving 306 Gy in two equivalent split doses is approximately 0.77. Even if damage induced by the first of the split doses was completely repaired, it might be expected that the survival would be near the level of the second dose alone, or near 0.15. Instead the survival is approximately five times greater, suggesting that the first split dose stimulated a repair system not present in unirradiated cells. The situation for confluent-holding recovery is similar to that for split-dose recovery.

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137 Cs γ rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.

TN-368 lepidopteran insect cells display a multiphasic survival response in both air and nitrogen. In each case the survival curve is characterized by an initial small-shouldered component having a steep slope, a plateau or broad-shouldered region near the 0.1 survival level, and finally a shallow slope component. The D 0 , <tex-math>$D_{{\rm q}}$</tex-math>, and n values for the initial steep slope component in air and nitrogen are, respectively, 65.7 Gy, 9.0 Gy, and 1.2, and 104.4 Gy, 28.8 Gy, and 1.3. The oxygen enhancement ratio (OER) for this portion of the curve is 1.6. The D 0 , <tex-math>$D_{{\rm q}}$</tex-math>, and n values for the shallow slope component in air and nitrogen are, respectively, 130.2 Gy, -36.1 Gy, and 0.8, and 226.8 Gy, 121.0 Gy, and 1.7. The OER for this portion of the curve is 1.7. The D 0 values for each slope and the width of the plateau region all increase proportionally for the nitrogen curve over that of air, the OER being approximately the same for both curve components. A similar multiphasic response was observed at dose rates of 202, 49.6, and 9.1 Gy/min. In addition, the survival of cells which had previously been irradiated with a dose well into the logarithmic region of the more resistant shallow slope portion of the curve retained a multiphasic response. Although cell cycle variations in radiosensitivity may contribute slightly to the response, an inducible or activated repair process would be consistent with the results.

The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CH1, clone GV1, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with <tex-math>$D_{0},\ D_{{\rm q}}$</tex-math>, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D 0 of 63.9 Gy, <tex-math>$D_{{\rm q}}$</tex-math> of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects.

The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells. D 0 values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

Cultured Trichoplusia ni cells in exponential growth were administered X-ray doses of 10,000 R and then subcultured. The concentration of alanine increased greatly and ammonia increased slightly in media of both untreated and irradiated cells up to 96 hr. Glycine concentration did not change significantly while the concentration of the remaining amino acids decreased to varying extents in media from both irradiated and untreated cultures. It was assumed that a decreasing amino acid concentration in the growth medium was due to utilization of the amino acids by the cells while an increasing amino acid concentration reflected its production by the cells. In medium from both untreated and X-irradiated cultures, a decrease in glutamic acid concentration was proportional to an increase in alanine concentration. With time spent in culture, the amounts consumed and produced per cell were much higher in the irradiated cultures.

Cultured Trichoplusia ni cells in exponential growth were administered X-ray doses of 10,000 R and then subcultured. The untreated cell population began exponential growth within a few hours after subculture, eventually reaching stationary growth phase 96 hr later at a cell density of <tex-math>$2.08\times 10^{6}$</tex-math> cells/ml, whereas the irradiated cell population did not change for 24 hr after irradiation and then began exponential growth at a rate similar to that of control cells, also reaching stationary phase at 96 hr, but at a cell density of <tex-math>$0.93\times 10^{6}$</tex-math> cells/ml, which is less than half the maximum density of controls. From 24 to 96 hr after treatment, the X-irradiated cells were characterized by an increased consumption of oxygen that was nearly twice the amount utilized by control cells. The pH of the cell growth medium increases over 96 hr from 6.3 to 6.6 for irradiated as well as for untreated cultures, but since the number of X-rayed cells is less than half the number of untreated cells, the pH increase, per cell, of medium from irradiated cultures is about twice that of medium from control cultures.