Potentially Lethal Damage Recovery Research Articles

Potentially lethal damage repair in drug arrested G2-phase cells after radiation exposure.

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2.

Differential influence of TGFβ1 and TGFβ3 isoforms on cell cycle kinetics and postirradiation recovery of normal and malignant colorectal epithelial cells

A clonogenic assay was used to determine the effects of the growth factors TGFbeta1 and TGFbeta3 on the radiation responses of a normal rat epithelial cell line (IEC6) and a human colonic carcinoma epithelial cell line (Widr). The radiation sensitivity and ability to recover from potentially lethal damage (PLD), of preconfluent monolayer cultures, was assessed in the presence of the growth factors for 24 h prior to, during, and after irradiation. The surviving fractions of both cell lines assessed immediately following irradiation were unaffected by TGFbeta1 or TGFbeta3. However, TGFbeta3 (but not TGFbeta1) significantly reduced the amount of PLD recovery in the Widr cells (but not in the IEC6 cells). This was associated with a reduction in the shoulder region of the survival curve, rather than a change in slope. A comparative analysis of the effects of TGFbetas 1 and 3 on cell cycle events in the two cell lines demonstrated significantly more Widr cells in the S phase, in the presence of TGFbeta3 only, compared to the controls. This remained constant both before and immediately following irradiation. In the IEC6 cell line TGFbeta3 produced an increase in the numbers of G1 phase cells, characteristic of a G1 arrest. It seems likely that TGFbeta3-induced radiosensitisation in Widr cells, 6 h after a single dose of irradiation, is related to its effects on cell cycle events such that the failure of these cells to arrest in G1, either before or after irradiation, results in significantly reduced recovery from DNA damage. This, however, may not be the only mechanism by which this growth factor produces this effect. Indeed, it will also be necessary to investigate these effects in in vivo models and to determine the response to fractionated irradiation before the potential therapeutic benefit of both the differential effects observed between the two TGFbeta isoforms and also between the malignant and normal cell lines can be fully assessed.

Differences in repair of potentially lethal damage in Chinese hamster ovary cells exposed to 65 MeV proton beams and (137)Cesium gamma-rays

Our study aimed to establish whether there are differences in the repair of potentially lethal damage (PLD) resulting from exposure to 65 MeV proton beams and (137)Cesium (Cs-137) gamma-rays. Irradiation with proton beams was carried out at the plateau and Bra,og peak in unmodulated beams, and at the spread-out Bragg peak (SOBP) for modulated beams. After irradiation, Chinese hamster ovary (CHO) cells in plateau phase were either plated immediately or kept in their own spent medium at 37 degrees C for various times up to 8 hours before plating to dishes or microplates. Survival was assessed with two methods: clonogenic and metabolic activity (MTT) assay. Obvious repair was observed in response to both kinds of irradiation beams, including different irradiation positions in proton beams. The clonogenic assay showed the PLD recovery ratios of 2.05, 1.79, 1.66 and 1.32 at two hours after 5 Gy irradiation for Cs-137 gamma-rays, plateau, SOBP and Bragg peak of proton beams, respectively. Recovery ratios for the SOBP and Bragg peak were significantly lower than that for gamma-rays (p<0.05 or p<0.01). There were no significant differences in the recovery ratios assessed by different methods (clonogenic and MTT assay), except for the Bragg peak. PLD repairs for proton beams and Cs-137 gamma-rays were not found at lower dose irradiation (3 Gy). Our results indicate that PLD repair in response to 65 MeV proton beams (SOBP and Bragg peak) is less and occurs later than for Cs-137 gamma-rays. The MTT assay was found to be a useful method in radiobiological research of proton beams.

Inhibition of potentially lethal damage recovery by altered pH, glucose utilization and proliferation in plateau growth phase human glioma cells.

Recovery from potentially lethal damage (PLD) has been measured in plateau growth phase human glioblastoma cells (U-87MG) under four postirradiation medium conditions. Recovery was maximal in depleted medium at an acidic pH, conditions which inhibit cellular proliferation. Compared with this control, PLD recovery (PLDR) was increasingly inhibited by alkalization of the existing medium (to pH 7.4), exchanging the old medium with fresh medium-pH acidified (to pH 6.8), and exchanging the old medium with fresh medium-pH unaltered (pH 7.4), respectively. These three medium adjustments were made at the time of irradiation. Increased glucose utilization (glycolysis) was detected postirradiation in all three cases, while increased proliferation was detected only when fresh medium was exchanged for old medium. Thus inhibition of PLDR has been correlated with increased glycolysis and increased proliferation during the recovery period. When acting together, these two processes provided almost complete inhibition. This study was revealed that the degree of inhibition may be related to the amount of glycolysis and/or proliferation occurring during the recovery period. Examining, in vitro, the range of PLDR achieved by postirradiation manipulation of medium pH may provide some indication of the range in PLDR that may be expected in vivo. Our study demonstrates that the effect of pH on glycolysis and proliferation may be important when determining the ability of a particular cell type to recover from PLD.

Inhibition of recovery from potentially lethal radiation damage in A549 cells by the [formula omitted] ionophore nigericin

A549 cells held for 4 hr in Hank's balanced salt solution, after 10 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is dependent on extracellular pH (pHe). Recovery factors of 2.2 to 3.5 are observed when pHe is 6.40 to 7.30, but recovery factors of less than 1.0 are found when pHe is reduced to 6.20 or 6.00. The K + H + ionophore nigericin, when added to cells post-irradiation, inhibits PLDR in a pHe-dependent manner, it is increasingly more effective as pHe is reduced from 6.80 to 6.40. The presence of nigericin thus causes inhibition of PLDR at pHe's that normally promote recovery. The drug does not affect radiation response of A549 cells when present only during irradiation. Effects of low pHe buffer, with and without nigericin, on intracellular pH (pHi) and on ATP levels were examined in an effort to elucidate the mechanisms for inhibition of PLDR and enhancement of radiation response. Incubation of cells in pHe 6.00 buffer results in a slight decrease in pHi and does not induce a drop in ATP levels. In contrast, post-irradiation incubation of cells in pHe 6.40 buffer containing 2 γM nigericin causes an immediate and dramatic decrease in pHi, and a gradual loss of ATP to 30% of control levels by 4 hr. The data obtained so far suggest that a very slight lowering of pHi may influence post-irradiation holding recovery, and that the mechanisms by which pHe 6.00 buffer alone, or pHe 6.40 buffer containing nigericin, affect holding recovery are different.

Inhibitors of Repair of Potentially Lethal Damage and DNA Polymerases Also Influence the Recovery of Potentially Neoplastic Transforming Damage in C3H10T-1/2 Cells

The effects of aphidicolin and beta Ara A on radiation sensitivity were evaluated in terms of cell killing, recovery, and neoplastic transformation in the C3H10T-1/2 cell system. When cells were held in plateau phase, recovery of potentially lethal damage (PLD) and potentially transforming damage (PTD) occurred. The addition of beta Ara A resulted in reduced PLD recovery for both the survival and neoplastic transformation end points. The addition of aphidicolin did not affect recovery of PLD or PTD. These data show that the inhibition of polymerase alpha by aphidicolin does not affect recovery of damage leading to cell death or neoplastic transformation. However, the inhibition of both polymerase alpha and beta by beta Ara A resulted in inhibition of recovery of damage leading to both cell death and neoplastic transformation. These data indicated that polymerase beta may be involved in both PLD and PTD recovery.

PH-Dependent Effects of the Ionophore Nigericin on Response of Mammalian Cells to Radiation and Heat Treatment

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.

Lethal Effect and Potentially Lethal Damage Recovery in Cultured Mammalian Cells Irradiated by Neutron-capture Beams

Cell killing and potentially lethal damage (PLD) recovery in Hela cells were examined after irradiation in suspension using a 10B neutron-capture beam. Thermal-neutron irradiation in the medium containing from 0.09 to 9 mM boron-10 (0.9-90 micrograms/g 10B, 90 per cent enriched boric acid) resulted in steeper survival curves than in the boron-free medium. The relative biological effectiveness (r.b.e.), expressed as the Do ratio, increased from 2.2 +/- 0.1 for the control without boron-10 to 5.4 +/- 0.3 in the medium containing 0.9 mM boron-10. The r.b.e. value was 5.2 +/- 0.5 at a boron-10 concentration of 9 mM, and did not increase when the boron-10 concentration was increased from 0.9 to 9 mM. It was suggested that the observed plateau of r.b.e. at boron-10 concentrations higher than 0.9 mM was due to the dominancy of the 10B(n, alpha)7Li reaction for dose accumulation. PLD recovery after the neutron irradiation containing 0.9 mM boron-10 was not observed, while a small effect was observed after irradiation in the thermal neutron beam, although much less than after gamma rays. The results show that the neutron-capture beam is an excellent beam for radiotherapy if the boron-10 concentration in tumour cells could be elevated to about 1 mM.

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.

Reduced pH increases recovery from radiation damage potentially leading to cell death and to in vitro transformation.

C3H-10T1/2 cells were grown to plateau phase and assessed for recovery from radiation damage under acidic and normal pH conditions. Repair of potentially lethal damage (PLD) was observed after X-ray doses of 5.0, 6.0 and 12.0 Gy and repair increased with dose. The repair at an acidic pH of 6.8 was greater than at the normal pH of 7.4. Repair of potentially transforming damage (PTD) was also observed when cells were held in plateau phase after irradiation. Repair of PTD was greater at the acidic pH than at the normal pH. When PTD recovery was plotted vs PLD recovery the results showed a good linear correlation with approximately twice as much PTDR. The experimental conditions allowing PLDR and PTDR indicate that the repair process may be error-free, since transformation was reduced in all experiments which showed recovery from PLD.

The Effect of pH on Potentially Lethal Damage Recovery in A549 Cells

The radiation sensitivity and potentially lethal damage recovery (PLDR) capacity of A549 human lung carcinoma cells have been studied. For unfed monolayer cultures, radiation sensitivity was greater in plateau phase than in log phase of growth. PLDR was observed when plateau-phase cells were held in their own spent medium postirradiation, such that the dose-response curve with 24 h holding was similar to that for log-phase cells plated immediately after irradiation. The high PLDR capacity of A549 plateau-phase cells (recovery factor between 40 and 70 for 24 h holding after 10 Gy) was reduced 10-fold or more by alkalinizing the pH of the spent medium immediately after irradiation from a value of 6.5 +/- 0.1 to a value of 7.6. Medium alkalinization resulted in an increase in the rate of glycolysis, with subsequent reacidification to a pH of 7.3 within 2 h of the pH adjustment. No change in cell cycle distribution was observed in the plateau-phase cultures up to 32 h after change of medium pH, and no increase in cell density was found after 48 h. A slight increase in the rate of incorporation of radiolabeled thymidine into acid-precipitable material was observed at 4 and 24 h after alkalinization of the medium. While it is not possible at present to define a mechanism for this pH effect, our results demonstrate that, at least for this cell line, variables such as medium pH and glucose concentration can profoundly influence the observation of PLDR.

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.

Influence of inhibitors of poly(ADP-ribose)polymerase activity on X-ray and ultraviolet light induced killing and mutation in Chinese hamster V79 cells.

The effect of nicotinamide or benzamide, inhibitors of poly(ADP-ribose)polymerase activity, on X-ray and ultraviolet light induced killing of asynchronously dividing V79 Chinese hamster cells was to reduce the shoulder width of the survival curve; the rate of killing remaining unchanged and the yield of 8-azaguanine resistant mutants also remaining unaffected. However, when density inhibited cells were irradiated with X-rays or u.v. light, holding at this density allowed potentially lethal damage recovery (PLDR) to occur, enhancing survival. Inhibitors of poly(ADP-ribose)polymerase suppressed PLDR and also increased the yield of mutants. Thus the role of poly(ADP-ribose) in X-ray or u.v. induced killing and mutation was dependent on the condition of growth.

Effect of Hypoxia on Recovery from Damage Induced by Heat and Radiation in Plateau-Phase CHO Cells

The effect of hypoxia on the induction of and recovery from damage by radiation alone and in combination with heat has been investigated using plateau-phase Chinese hamster ovary (CHO) cells. Postirradiation hypoxia reduced the potentially lethal damage recovery (PLDR) in cells irradiated under an euoxic state and completely eliminated PLDR in cells irradiated under hypoxia. Cells which were maintained under hypoxia during both irradiation and a 4-hr recovery period and then incubated for a further period of 4 hr under euoxic conditions showed PLDR, suggesting that the inhibition of PLDR by hypoxia is reversible. Oligomycin, an inhibitor of energy metabolism, completely eliminated PLDR when present at a concentration of 1 microM during the postirradiation period. Pre- or postirradiation heat treatment at 42.5 degrees C for 30 min appreciably sensitized the cells to the induction of lethality. Thermal enhancement ratio (TER) was 1.7 for cells irradiated and heat treated under hypoxic conditions. The same heat treatment reduced the oxygen enhancement ratio (OER) associated with gamma radiation from 3.1 to 2.5. Cells subjected to this postirradiation heat treatment showed a small extent of PLDR, whereas the pre-heat-treated cells showed as much recovery as non-heat-treated cells. When hypoxic conditions prevailed during the post-treatment incubation period, PLDR was reduced in preheated cells and completely eliminated in postheated cells. The kinetics of interaction between heat and radiation damage were studied by introducing a time gap of 4 hr between the treatments. Cells maintained under euoxic conditions between the treatments showed an appreciable decrease in interaction, suggesting recovery from damage induced by the first treatment. Hypoxic conditions intervening the two treatments largely inhibited the loss of sensitization. Analysis of the results suggests that cells fail to recover from sublethal heat damage when held for 4 hr under hypoxic conditions. Cells held under hypoxic conditions partly recover from the radiation damage which subsequently interacts with sublethal heat damage, resulting in cell lethality.

The role in cancer therapy of inhibiting recovery from PLD induced by radiation or bleomycin

Effects on survival of allowing and inhibiting potentially lethal damage recovery (PLDR) after X ray or bleomycin (bleo) exposure, especially at clinically applicable doses, were examined in plateau phase Chinese hamster ovary (CHO) cells. Dose modifying factors (DMFs) between immediately explanted cells and those trypsinized 24 hours after various doses of X rays were 2.51 t 0.12 (mean ± 2 S.E.) at 70% surviving fraction (SF), 2.03 ± 0.085 at 40% and 1.71-1.79 at lower SF levels. Thus, at doses used clinically (1 – 4 Gy), the DMF by PLDR was more than 2.0, suggesting its importance in radiotherapy of cancers. Feeder layers were found to increase the SF, especially for replating immediately after bleo exposure. Among the inhibitors tested, 3′-deoxyguanosine at 3.7 mM and caffeine at 10.3 mM inhibited x and bleo PLDR (DMF of 1.0–1.2) when trypsinized 24 hours after treatment. However, interestingly, 3 aminobenzamide, a specific inhibitor of poly (ADP) ribose synthesis, even at 14.7 or 29.4 mM, was not as effective in suppressing both x and bleo PLDR, suggesting the role of repair processes independent of poly (ADP) ribose levels in PLDR in plateau phase cultures. Possibilities of clinical application of PLDR inhibitors as radio- and chemosensitizers are discussed.

Polar solvent modification of X ray induced potentially lethal damage in heterogeneous human colon tumor cells in vitro

Two subpopulations of tumor cells (clones A and D) obtained from a human colon adenocarcinoma were examined for their sensitivities to x-irradiation as unfed, early plateau phase cultures. Both the single dose survival curves and the kinetics of potentially lethal damage recovery (PLDR) were determined for the two tumor lines. Also, possible modification of PLDR by N,N-dimethylformamide (DMF ), which has previously been shown to enhance the radiosensitivity of exponentially growing tumor cells, was investigated by adding DMF (0.896 v/v) to plateau phase cultures immediately after irradiation, and determining effects on the extent of PLDR. For non-DMF treated cells, the survival curve parameters of the diploid (clone D) and aneuploid (clone A) lines were very similar. The single-hit, multitarget values for n, D 0 (Gy), and D, (Gy) were: 7.9, 0.82, and 1.70 for clone D; and 10.6, 0.83, and 1.96 for clone A. Using initial survival levels of 3.5% (clone D) or 5.5% (clone A) to investigate PLDR, it was found that the increase in survival (surviving fraction ratio or SFR) for clone D was 2.2, while the SFR for clone A was 1.6. DMF did not change either the kinetics or extent of PLDR in these two tumor lines when added to cultures immediately after irradiation. Our results indicate that significant heterogeneity in PLDR exists between these closely related tumor subpopulations.

Repair of Potentially Lethal Damage in Chinese Hamster Cells after X and α Irradiation

Recovery from potentially lethal damage (PLD) of cultured Chinese hamster cells (line CHO) after exposure to x rays and to ..cap alpha.. particles was investigated using both exponentially growing and plateau-phase cells (nutritionally induced stationary phase). The results indicated that there was no recovery from PLD after exposure to ..cap alpha.. particles from plutonium. Experiments were also conducted to determine if recovery phenomena were dependent on cell concentration. Recovery from PLD after x-ray exposure was not dependent on cell concentration. Cell-to-cell contact also does not seem to be an important factor in PLD recovery. The recovery curve shape after exposure to x rays, both for exponentially growing and plateau-phase CHO cells, remained identical; however, the magnitude of repair by plateau-phase cells was higher than by exponentially growing cells, indicating that cells in G/sub 1/ may be primarily involved in PLD recovery.