Independent forms of potentially lethal damage fixed in plateau-phase Chinese hamster cells by postirradiation treatment in hypertonic salt solution or araA

Plateau-phase Chinese V79 hamster cells were sequentially treated after exposure to gamma rays in medium made hypertonic by the addition of sodium chloride (370 mM) and with various concentrations of 9-beta-D-arabinofuranosyladenine (araA) to study their combined effect on fixation of potentially lethal damage (PLD). A 10-min treatment in hypertonic medium fixed an extensive amount of PLD and caused a decrease in D0 from 1.8 to 1.2 Gy without significantly affecting Dq. Subsequent treatment with araA caused further fixation of PLD but resulted in a specific, concentration-dependent reduction in Dq from 4.9 to 1.6 Gy after a 4-h exposure to 150 microM araA. A 30-min treatment in hypertonic medium reduced not only Do (from 1.8 to 1.0 Gy) but also Dq (from 4.9 to 2.7 Gy). Subsequent treatment with araA in this case affected only the residual shoulder, reducing it to 1.6 Gy after a 4-h treatment with 100 microM araA, a value similar to that obtained after treatment with araA of cells exposed to salt for only 10 min. When the repair of PLD fixed by a 10-min treatment with salt was measured by delaying its postirradiation application in the presence of various amounts of araA, a small decrease in the repair rate was observed but no significant effect on the relative increase in survival. Qualitatively similar results were obtained for repair of PLD sensitive to araA after a 10-min treatment in hypertonic medium. These results suggest the radiation induction of forms of PLD with different sensitivity to fixation by postirradiation treatments. araA is proposed to fix a form of PLD termed alpha-PLD, the repair of which takes place within 4-6 h and which causes the formation of the shoulder in the survival curve of cells plated immediately after irradiation. Short treatments in hypertonic medium (less than 10 min) are proposed to fix a form of PLD termed beta-PLD, the repair of which takes place within 1 h and leads to restoration of the slope to values equal to those obtained in the survival curve of cells plated immediately after irradiation. However, longer treatments in hypertonic medium also affect Dq and thus also alpha-PLD. Repair of beta-PLD was not significantly affected by araA and repair of alpha-PLD was not significantly affected by short hypertonic treatment, thus indicating the independence of the two forms of PLD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Evidence for the induction of two types of potentially lethal damage after exposure of plateau phase Chinese hamster V79 cells toγ-rays

Summary The fixation of-rays induced potentially damage (PLD) caused after treatment either with-araA or in medium made hypertonic by the addition of sodium chloride was studied in plateau phase chinese hamster V79 cells. Treatment with-araA was found to affect a sector of PLD, the fixation of which specifically reduced the shoulder width of the survival curve. The effect was maximized when cell survival reached levels corresponding to an exponential line, with a slope similar to the final slope of the survival curve of untreated cells. This effect was achieved by a four hour treatment with-araA at concentrations above 150µM. Longer treatment times or incubation at higher-araA concentrations did not significantly enhance the effect. Treatment in hypertonic medium, on the other hand, enhanced cell killing in a concentration dependent (NaCl-concentration) way and the survival reached values much lower than those corresponding to an exponential line. No indication for a plateau in the effect, indicating complete fixation of the sector of PLD that reacts sensitively to this treatment, was obtained. Both the slope and the shoulder width of the survival curve were affected, the slope first being increased after short treatment times (up to 10 min), followed by a decrease in the shoulder width after longer treatment times (longer than 10 min). Lesions fixed after treatment with-araA were repaired within four hours, whereas the repair of lesions fixed after treatment in hypertonic medium (460 mM NaCl, 30 min) appeared to be biphasic, with a fast component (completed in about one hour) correlated with a decrease in the slope and a slow component (completed in four hours) correlated with restoration of the shoulder width. Based on these results, we suggest that two types of PLD may be induced in plateau phase V79 cells after exposure to-rays. One, the repair of which is completed within about one hour and which affects the slope of the survival curve, and a second, the repair of which takes place in a few hours and which specifically affects the survival curve shoulder width. The terms-PLD and-PLD are suggested for the first and second component, respectively.Comparison of the repair rates of-PLD as measured with the help of-araA and of sublethal damage as measured in split-dose experiments indicated that these two cellular repair processes have very similar kinetics when measured under the same experimental conditions. Furthermore, the rate was identical at which the shoulder of the survival curve reappeared (shoulder width was the only parameter of the survival curve affected in this type of experiment) in the time interval between either a conditioning dose of-rays and subsequent graded doses or between irradiation and treatment with-araA. Based on these results it is suggested that-PLD and sublethal damage may have a common molecular base.This work was supported by PHS-grants number CA 33951 and CA 39938 awarded by NCI, DHHS     

Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions.

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in DO. Fixation of radiation damage at 37 degrees C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 degrees C than at 20 degrees C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation.     

Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.     

Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.     

Interaction function gamma(x) for Chinese hamster cells treated with hypertonic phosphate-buffered saline after irradiation

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

Cell survival and recovery processes in Chinese hamster AA8 cells and in two radiosensitive clones

Cell survival and recovery after gamma irradiation were investigated in a Chinese hamster ovary cell line (AA8) and in two radiosensitive clones (EM9 and NM2) derived from it. When analyzed by the multitarget and linear-quadratic equations, the dose-response curves for survival of both EM9 and NM2 cells, compared with AA8 cells, were characterized by a decreased magnitude of the shoulder or single-hit region (as reflected by Dq or alpha, respectively) but no difference in the terminal slope or double-hit region (as reflected by DO or beta, respectively). Recovery from sublethal damage (SLD) and potentially lethal damage (PLD) was measured in the three cell lines to examine the relationship between the shoulder width of the survival curve and the magnitude of cellular recovery. NM2 cells exhibited a reduced shoulder on their survival curve and a reduced capacity for SLD recovery, compared with AA8 cells, after equitoxic doses of radiation. EM9 cells, which also had a reduced shoulder on their survival curve, displayed the same rate and extent of recovery as AA8 cells for both SLD and PLD. PLD recovery, as assayed in fed plateau-phase NM2 cells by delayed plating, occurred with slower initial kinetics but to the same final extent as that in AA8 cells, resulting in modification of both the shoulder and the slope of the survival curve. However, PLD recovery, as assayed in log-phase NM2 cells by postirradiation treatment with hypertonic salt, was normal and affected predominantly the slope of the survival curve. These data demonstrate that although both SLD and PLD recovery play a role in determining cell survival, cell-survival curve parameters may not always be useful in predicting cellular recovery capacity.     

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.     

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

Relationship between DNA repair and cell recovery: Importance of competing biochemical and metabolic processes

Summary The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were-irradiated with 1500 rad and then immediately treated for 20 min with a 37° C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. Hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation isotonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damageper se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation

Results are reported of studies to measure the extent of recovery of potentially lethal damage (PLD) in rat rhabdomyosarcoma tumor cells after irradiation both in vivo and in vitro with either high-LET or low-LET radiation. Stationary-phase cultures were found to exhibit repair of PLD following irradiation in vitro either with low-LET X rays or with high-LET neon ions in the extended-peak ionization region. Following a 9-Gy dose of 225-kVp X rays or a 3.5-Gy dose of peak neon ions, both of which reduced the initial cell survival to 6-8%, the maximum PLD recovery factors were 3.4 and 1.6, respectively. In contrast, the standard tumor excision assay procedure failed to reveal any recovery from PLD in tumors irradiated in situ with either X rays or peak neon ions. PLD repair by the in vivo tumor cells could be observed, however, when the excision assay procedure was altered by the addition of a known PLD repair inhibitor beta-arabinofuranosyladenine (beta-ara-A). When a noncytotoxic 50 microM concentration of beta-ara-A was added to the excised tumor cells immediately following a 14.5-Gy in situ dose of X rays, cell survival in the inhibitor-treated cells was lower than in the untreated cells (0.018 compared to 0.056), resulting in a PLD repair inhibition factor of 3.1. Delaying the addition of beta-ara-A for 1, 2, or 3 h following tumor excision reduced the PLD repair inhibition factor to 1.6, 1.5, and 0.9, respectively. Following tumor irradiation in situ with neon ions in the extended-peak ionization region (median LET = 145 keV/micron), less PLD repair was observed than after X irradiation. For 5.8 Gy of peak neon ions, the PLD repair inhibition factors were 2.1, 1.5, 1.3, and 1.1 at 0, 1, 2, and 3 h, respectively. We interpret the absence of measurable PLD repair using the standard tumor excision assay procedure as resulting from undetectable repair occurring during the long interval (about 2 h) required for the cell dissociation and plating procedures. We conclude that at least for our tumor system, PLD repair does occur after irradiation of tumors in situ, even though it is not detectable using the standard tumor excision assay procedure. Thus a failure to measure such repair by this assay in a given tumor system does not necessarily mean the cells are incapable of PLD repair.     

Sublethal and potentially lethal damage repair on thermal neutron capture therapy

Tonicity shock or caffeine postirradiation treatment makes evident fast-type potentially lethal damage (PLD). Caffeine expresses fast-type PLD more efficiently than tonicity shock in X-irradiated B-16 mouse melanoma cells, compared with V79 Chinese hamster cells. The survival curves of thermal neutrons for either V79 or B-16 cells exhibit no shoulder. Neither V79 nor B-16 cells show the sublethal damage (SLD) repair of thermal neutrons. Caffeine-sensitive fast-type PLD repairs exist in X-irradiated B-16 cells, as well as V79 cells. The fast-type PLD repair of B-16 cells exposed to thermal neutrons alone is rather less than that of X-irradiated cells. Furthermore, an extremely low level of fast-type PLD repair of B-16 cells with 10B1-paraboronophenylalanine (BPA) preincubation (20 hours) followed by thermal neutron irradiation indicated that 10B(n,alpha)7Li reaction effectively eradicates actively growing melanoma cells. The plateau-phase B-16 cells are well able to repair the slow-type PLD of X-rays. However, cells can not repair the slow-type PLD induced by thermal neutron irradiation with or without 10B1-BPA preincubation. These results suggest that thermal neutron capture therapy can effectively kill radioresistant melanoma cells in both proliferating and quiescent phases.     

Effects of adenosine deaminase inhibitor 2′-deoxycoformycin on the repair and expression of potentially lethal damage sensitive toβ-areaA

Summary The effects of 2deoxycoformycin, a specific inhibitor of adenosine deaminase, on the repair and fixation of potentially lethal damage (PLD) sensitive to treatment with-araA, an adenosine analogue acting via inhibition of DNA polymerases and, have been studied. Given after irradiation deoxycoformycin alone had little effect on cell survival. More damage was nevertheless fixed by a given concentration of-araA in the presence of deoxycoformycin to a degree that 35 µM-araA given simultaneously with 1 µg/ml deoxycoformycin produced the same effect (survival decrease to 20% of the controls) as 90 µM-araA given alone. Maximum potentiation of the-araA effect was observed at 1 µg/ml deoxycoformycin with a slight decrease at higher concentrations.Combined treatment with-araA and deoxycoformycin reduced or eliminated the shoulder from the survival curve without affecting the slope, an effect similar to that observed after treatment of cells with-araA alone. The results indicate the importance of adenosine deaminase in the inactivation of nucleoside analogues and are discussed vis-a-vis the possible practical application of this inhibitor in both experimental and therapeutic applications.     

Evidence for the repair of potentially lethal damage in irradiated bone marrow

Summary The effects on cell survival of maintaining bone marrow cells (CFU-S) in situ following irradiation and before assay by transplantation was investigated. When the CFU-S cells are maintained in situ following irradiation survival drops and plateaus at about 9 h post-irradiation. Evidence is presented that this decrease in survival may be due to potentially lethal damage repair (PLD) inhibition caused by post-irradiation in situ holding. This effect on PLD repair is different than that usually found in cells in vitro and in vivo tumors in that it mainly alters the shoulder rather than the slope of the survival curve of CFU-S cells. It is different than PLDR found in vivo for normal mammary and thyroid gland epithelial cells because in situ holding decreases rather than increases the survival of CFU-S cells. Evidence is also presented that the radiation survival curve for in situ bone marrow cells (CFU-S) may not have a shoulder.Supported in part by NIH, NCI grants P01 CA 19298 and P30 CA 14520Supported in part by an American Cancer Society Clinical Fellowship     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)