Potentially Lethal Damage Research Articles

Potential lethal damage repair in glioblastoma cells irradiated with ion beams of various types and levels of linear energy transfer.

Glioblastoma (GBM), a Grade IV brain tumour, is a well-known radioresistant cancer. To investigate one of the causes of radioresistance, we studied the capacity for potential lethal damage repair (PLDR) of three altered strains of GBM: T98G, U87 and LN18, irradiated with various ions and various levels of linear energy transfer (LET). The GBM cells were exposed to 12C and 28Si ion beams with LETs of 55, 100 and 200 keV/μm, and with X-ray beams of 1.7 keV/μm. Mono-energetic 12C ions and 28Si ions were generated by the Heavy Ion Medical Accelerator at the National Institute of Radiological Science, Chiba, Japan. Clonogenic assays were used to determine cell inactivation. The ability of the cells to repair potential lethal damage was demonstrated by allowing one identical set of irradiated cells to repair for 24 h before subplating. The results show there is definite PLDR with X-rays, some evidence of PLDR at 55 keV/μm, and minimal PLDR at 100 keV/μm. There is no observable PLDR at 200 keV/μm. This is the first study, to the authors’ knowledge, demonstrating the capability of GBM cells to repair potential lethal damage following charged ion irradiations. It is concluded that a GBM’s PLDR is dependent on LET, dose and GBM strain; and the more radioresistant the cell strain, the greater the PLDR.

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.

A H2S-Nampt dependent energetic circuit is critical to survival and cytoprotection from damage in cancer cells.

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.

DNA-PKcs Inhibition Sensitizes Cancer Cells to Carbon-Ion Irradiation via Telomere Capping Disruption

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.

Comparative Study of Radioresistance between Feline Cells and Human Cells

Radioresistance of cats has been seen in animal radiotherapy. Feline radioresistance and its underlying mechanism(s) were investigated in fibroblast cells and lymphocytes. We hypothesized that radioresistance was attributable to an increase in the cells ability to repair DNA damage. To investigate this hypothesis, fibroblast cells were exposed to various doses of X rays and then colony formation assays were performed. Survival curves showed that potential lethal damage repair (PLDR) for feline cells were greater than that for human cells. γ-H2AX foci assays were performed to evaluate DNA double-strand breaks (DSBs) formation and repair kinetics. After PLDR, feline cells displayed a decreased residual amount of γ-H2AX foci. Formation of chromosome aberrations (dicentrics) after PLDR as an indicator of radiation-induced DNA damage and repair; human, feline and canine lymphocytes were evaluated. Human and canine lymphocytes showed two to three times the number of dicentrics compared to feline lymphocytes. Finally, micronuclei assays were performed to further confirm the radioresistant nature of feline lymphocytes. In concordance with the results of the chromosome aberration assay, the number of micronuclei in feline lymphocytes was less than observed in human and canine lymphocytes. Taken together, these results show that DNA and chromosome damage induced by X irradiation is more effectively repaired in feline cells, resulting in less residual damage. Our results suggest that both feline fibroblasts and lymphocytes are more radioresistant compared to human cells of similar tissues, and this resistance can be contributed, at least in part, to greater ability for PLDR.

Comparison of the repair of potentially lethal damage after low- and high-LET radiation exposure, assessed from the kinetics and fidelity of chromosome rejoining in normal human fibroblasts

Potentially lethal damage (PLD) and its repair (PLDR) were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining after X-ray or heavy-ion exposures. Cells were either held in the non-cycling G0 phase of the cell cycle for 12 h, or forced to proliferate immediately after irradiation. Fusion premature chromosome condensation (PCC) was combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. The culture condition had no impact on the rejoining kinetics of PCC breaks during the 12 h after X-ray or heavy-ion irradiation. However, 12 h after X-ray and silicon irradiation, cycling cells had more chromosome exchanges than non-cycling cells. After 6 Gy X-rays, the yield of exchanges in cycling cells was 2.8 times higher than that in non-cycling cells, and after 2 Gy of 55 keV/μm silicon ions the yield of exchanges in cycling cells was twice that of non-cycling cells. In contrast, after exposure to 2 Gy 200-keV/μm or 440-keV/μm iron ions the yield of exchanges was similar in non-cycling and cycling cells. Since the majority of repair in G0/G1 occurs via the non-homologous end joining process (NHEJ), increased PLDR in X-ray and silicon-ion irradiated cells may result from improved cell cycle-specific rejoining fidelity through the NHEJ pathway, which is not the case in high-LET iron-ion irradiated cells.

The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: Evidence of hypoxia-induced potentially lethal damage

Purpose: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill.Materials and methods: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N2 gas containing 0, 0.1 or 0.5% O2. Cell survival was determined by colony formation assay immediately after 0–96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2–24 h before survival assay.Results: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O2 concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair.Conclusion: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.

Inactivation of ataxia telangiectasia mutated gene can increase intracellular reactive oxygen species levels and alter radiation-induced cell death pathways in human glioma cells

Purpose: To investigate the effects of ataxia telangiectasia mutated (ATM)-regulated reactive oxygen species (ROS) and cell death pathways on the response of U87MG glioma cells to ionising radiation (IR) and oxidative stress.Material and methods: ATM expression was blocked in U87MG glioma cells using a small interfering RNA (siRNA) technique. Cell survival, sub-lethal damage (SLD), and potential lethal damage (PLD) repair following IR were assessed by clonogenic assay while changes in intracellular ROS, the apoptosis, and autophagy were followed by flow cytometry and Western blotting.Results: Blocking ATM expression in U87MG cells increased intracellular ROS levels and sensitivity to the cytotoxic effects of IR and oxygen stress; effects that could be partly counteracted by the antioxidant N-acetylcysteine (NAC). Knock down of ATM rendered cells unable to repair sub-lethal or potentially lethal damage and DNA double strand breaks (DSB) after IR exposure; something that NAC could not counteract. ATM did control the pathways a cell used to die following IR and this did seem to be ROS-dependent.Conclusion: ATM is involved in redox control but ROS elevations following ATM knock down seem more involved in the decision as to what cell death pathway is utilised after IR than DSB repair and radiosensitivity.

A Comparison of Chromosome Repair Kinetics in G0and G1Reveals that Enhanced Repair Fidelity under Noncycling Conditions Accounts for Increased Potentially Lethal Damage Repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.

Mitigation of Irradiation Induced Potentially Lethal Damage (PLD) in Hematopoietic Cells by Mitochondrial Localized GS-Nitroxide, JP4-039

PLD is an ionizing irradiation pathway believed to apply to quiescent hematopoietic cells in the marrow microenvironment. To determine if the mitochondrial targeted JP4-039 which has been shown to mitigate sublethal irradiation damage (SLD) in non-quiescent clonagenic cells protected cells from PLD, 32D cl 3 hematopoietic cells were irradiated in three sets of cultures to doses ranging from 0 to 8 Gy. To one group of cells, to measure SLD mitigation effects, JP4-039 (10 μM) was added immediately, then cells plated in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. The second set of irradiated cells was grown in flasks in 10 ml of liquid media. The third set was tested for PLD effects and cells were centrifuged and maintained in pellets in 1 ml of media (PLD conditions preventing cell growth or division) and these latter two sets were kept at 37°C. After 24, 48 or 72 hrs irradiation, subsets of each were removed, viability and cell counts were determined and JP4-039 was then added to subgroups of cultured cells at each time point, cells re-suspended in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. Viability of cells in non-irradiated pellets decreased from 24 to 72 hours from 60.3 ± 2.7% to 37.3 ± 2.3% (p = 0.0030), while after 8 Gy the cell viability was 57. 3 ± 0.3% at 24 hr and 25.7 ± 6.1% at 72 hr (p = 0.0065). There was no significant decrease in percent viability at 24 hrs of non-irradiated or 8 Gy irradiated cells (60.3 ± 2.7% and 57.3 ± 0.3%, respectively) or at 72 hr (37.3 ± 2.3% and 25.7 ± 6.1%, respectively). In contrast, non-irradiated cells re-suspended in flasks grew and thus viability increased from 77.0 ± 4.0% at 24 hr to 96.7 ± 0.3% at 72 hr (p = 0.0080). Cell viability after 8 Gy of surviving irradiated cells grown in flasks was not significantly changed from 64.0 ± 10.6% at 24 hr to 49.3 + 3.2% at 72 hr. There was no significant change in cell viability in flasks at 24 hrs of non-irradiated or 8 Gy irradiated cells (77 ± 4.0% and 64.0 ± 10.6%, respectively). Cell viability was significantly decreased at 72 hr in non-irradiated cell flasks of 96.7 ± 0.3% compared to 8 Gy irradiated flasks with a viability of 49.3 ± 3.2% (p = 0.0001). Cells maintained in pellets showed there was no cell growth between 24 to 72 hours while those maintained in flasks did significantly grow for non-irradiated cells from 6.3 ± 0.2 × 105 cells at 24 hr to 85.3 ± 6.8 × 105 cells at 72 hr (p = 0.0003). For 8 Gy irradiated cells grown in flasks, there was no significant change in the number of cells between 24 and 72 hr (4.9 ± 0.9 × 105 and 4.9 ± 1.2 × 105, respectively). Thus, PLD conditions were established in pellets. Irradiation survival curves showed a JP4-039 mediated PLD radiation resistance when cells were incubated in JP4-039 at 24 hr or 72 hr after irradiation by increase in the Do. At 24 hr, the Do for pelleted cells incubated in JP4-039 was 1.92 ± 0.01 Gy compared to pelleted irradiated control 32D cl 3 cells, 1.36 ± 0.02 Gy (p = 0.0014). At 24 hrs, cells grown in flasks and incubated in JP4-039 had a Do of 1.70 ± 0.02 Gy compared to 1.40 ± 0.05 Gy for irradiated control 32D cl 3 cells (p = 0.0261). By 72 hrs after irradiation, the Do for pelleted cells was increased to 1.97 ± 0.06 Gy by adding JP4-039 compared to the pelleted irradiated control 32D cl 3 cells with a Do of 1.51 ± 0.04 Gy (p = 0.0218). In cells grown in flasks for 72 hr before the addition of JP4-039 the Do was 2.01 ± 0.02 Gy compared to irradiated controls which had a Do of 1.62 ± 0.07 Gy (p = 0.0346). Therefore, JP4-039 displays a clear potential to mitigate irradiation induced PLD damage. These data correlate with significant mitigation of the hematopoietic syndrome by JP4-039 in total body irradiated C57BL/6NHsd mice.

Inhibition of a Radiation-Induced Senescence-Like Phenotype: A Possible Mechanism for Potentially Lethal Damage Repair in Vascular Endothelial Cells

The well-established process of potentially lethal damage (PLD) repair enhances plateau-phase cell survival after exposure to ionizing radiation. PLD repair requires that confluent cells be incubated prior to plating for a colony-forming assay rather than being plated immediately. Enhanced double-strand break (DSB) repair during this incubation period has been implicated in the enhanced survival, but the precise molecular mechanism and its biological significance remain largely unclear. Radiation has been recently reported to induce premature senescence, and increasing evidence suggests that DSBs commonly mediate cellular senescence. Here we successfully related these two biological phenomena using bovine aortic endothelial cells, and propose that enhanced DSB repair during the plateau-phase incubation prevents expression of the radiation-induced senescence-like phenotype, eventually leading to an enhanced colony-forming ability. This could be a novel biological interpretation of PLD repair.

G0Cell Cycle Arrest Alone is Insufficient for Enabling the Repair of Ionizing Radiation-Induced Potentially Lethal Damage

The repair of ionizing radiation-induced potentially lethal damage (PLD) is suggested to be important for the clinical response to radiotherapy. PLD repair is usually studied in quiescent cultures prepared by growing cells to confluence with an accumulation of cells in G(0) phase of the cell cycle, but the biological pathways enabling PLD repair are still unknown. In this study, we examined whether the controlled expression of two different inducers of G(0) cell cycle arrest, the human tumor suppressor gene growth arrest specific 1 (GAS1) in murine fibroblasts and the forkhead transcription factor FOXO3a in human colon carcinoma cells, is sufficient to enable PLD repair. We found that GAS1 and FOXO3a induced a cell cycle arrest in G(0) phase with a concomitant reduction of proliferation of log-phase cells. In both cell systems, this cell cycle arrest in G(0) phase did not enable PLD repair in log-phase cells. Significant PLD repair was found in all confluent cultures that showed similar cell cycle distributions, while GAS1 and FOXO3a in confluent cells did not influence PLD repair. No differences were found in cell cycle re-entry after replating cells with different capacities for PLD repair. Our data suggest that the induction of G(0) cell cycle arrest and the reduction of proliferation are not sufficient to enable PLD repair.

Inhibition of Potential Lethal Damage Repair and Related Gene Expression after Carbon-ion Beam Irradiation to Human Lung Cancer Grown in Nude Mice

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we previously demonstrated their radiosensitivity by focusing attention on the dynamics of tumor clonogens and the early and rapid survival recovery (potential lethal damage repair: PLD repair) occurring after X-ray irradiation. To the authors' knowledge, this is the first study demonstrating gene expression in association with PLD repair after carbon-ion beam or X-ray irradiation to cancer cells. In this study we tried to detect the mechanism of DNA damage and repair of the clonogens after X-ray or carbon-ion beam irradiation. At first, colony assay method was performed after irradiation of 12 Gy of X-ray or 5 Gy of carbon-ion beam to compare the time dependent cell survival of the IA cells after each irradiation pass. Second, to search the genes causing PLD repair after irradiation of X-ray or carbon-ion beam, we evaluated gene expressions by using semi-quantitative RT-PCR with the selected 34 genes reportedly related to DNA repair. The intervals from the irradiation were 0, 6, 12 and 24 hr for colony assay method, and 0, 3, 18 hr for RT-PCR method. From the result of survival assays, significant PLD repair was not observed in carbon-ion beam as compared to X-ray irradiation. The results of RT-PCR were as follows. The gene showing significantly higher expressions after X-ray irradiation than after carbon-ion beam irradiation was PCNA. The genes showing significantly lower expressions after X-ray irradiation rather than after carbon-ion beam irradiation were RAD50, BRCA1, MRE11A, XRCC3, CHEK1, MLH1, CCNB1, CCNB2 and LIG4. We conclude that PCNA could be a likely candidate gene for PLD repair.

DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1–20 μM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 μM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2–3 folds at 5 μM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G 2 blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.

DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1–20 μM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 μM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2–3 folds at 5 μM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G 2 blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells.

The functionality of G(1)-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after gamma irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G(1) arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G(1) arrest.

Mechanism of potentially lethal damage (PLD) and its repair (PLDR) in non-growing G0 cells; high frequency of misrepair is induced under PLD condition

Mechanism of potentially lethal damage (PLD) and its repair (PLDR) in non-growing G0 cells; high frequency of misrepair is induced under PLD condition

Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation.

Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy gamma rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.

Comparison of repair and rejoining fidelity between two isogenic human ovarian carcinoma cell lines

Purpose : The difference in radiosensitivity between two isogenic tumour cell lines was evaluated to determine whether factors such as sublethal and potentially damage repair, DNA double-strand break repair and fidelity of repair can be related to differences in radiosensitivity. Materials and methods : The cell lines used were the ovarian carcinoma A2780s and a radiation-resistant derivative A2780cp. Radiation response was measured in terms of cell survival, recovery of sublethal (SLD) and potentially lethal damage (PLD), induction of and recovery of DNA strand breaks, and fidelity of DNA repair using a cell-free plasmid assay. Results : While A2780cp was more resistant to radiation than A2780s, it showed less ability for recovery of SLD and PLD. DNA strand-break induction was the same for both cell lines, while only at very high doses did A2780cp show greater DNA strand-break recovery than A2780s. Fidelity of rejoining DNA was significantly higher in the A2780cp cell line. Conclusion : The difference in radiosensitivity between these two cell lines was not related to recovery of PLD or SLD or to the induction of DNA damage. It appears that fidelity of DNA rejoining, which was significantly higher in the resistant cell line, may be related to the difference in radiosensitivity.

Importance of cell proliferative state and potentially lethal damage repair on radiation effectiveness: implications for combined tumor treatments (review).

The dependence of parameters of the linear-quadratic (LQ) model on cell proliferation kinetics of tumors in relation to potentially lethal damage (PLD) and its repair is evaluated. The influence of sensitizing agents on these parameters during fractionated radiotherapy is assessed. Suggestions for scheduling of radiation combined of with sensitizing agents are derived. The parameters alpha and beta of the linear-quadratic model for dose dependence of cell reproductive inactivation, derived from experimental and clinical data, are evaluated to assess their dependence on cell proliferative state, on PLD repair and on the action of various sensitizing agents. PLD contributes to the linear as well as to the quadratic component of the LQ model. PLD is less effectively repaired in proliferating (P) cells than in clonogenic (G0) cells of the quiescent (Q) cell compartment. PLD is influenced by various agents applied during, as well as after irradiation. The parameters alpha and beta are affected differently by the proliferative state of cells, by some of the sensitizing agents, and by radiation quality. The relative fractions of P cells and Q cells can change during fractionated treatments. If recruitment is effective, the fraction of G0 cells decreases in the latter part of a treatment schedule. PLD from subsequent radiation doses is then repaired less and the effectiveness of radiation combined with sensitizing agents may be enhanced. The analyses using the LQ model show differences in PLD and its repair between P cells and G0 cells in tumors. If due to recruitment the compartment of clonogenic G0 cells diminishes during treatment, the combination of radiation with sensitizing agents and the application of high-LET radiation should be scheduled to take this factor into account. For poorly differentiated tumors with high labeling indices (LI), benefit from combined treatments is expected from early in the course of fractionated radiotherapy. Well differentiated tumors with low LI are suggested to benefit most from irradiation combined with sensitizing agents in the latter part of a treatment schedule. New methods are required to assess the clonogenic G0 cells in the Q cell compartment and to monitor recruitment of these cells into the P cell compartment.