Potentially Lethal Damage Repair Research Articles

Palmitoyl ascorbic acid glucoside enhanced cell survival with post irradiation treatment

Radiation exposure poses a significant threat to cellular integrity by inducing DNA damage through the generation of free radicals and reactive oxygen species. Ascorbic acid, particularly its derivative Palmitoyl Ascorbic Acid 2-Glucoside (PA2G), has demonstrated remarkable radioprotective properties. While previous research focused on its pre-irradiation application, this study explores the post-irradiation radiomitigation potential of PA2G. Our findings reveal that post-irradiation treatment with PA2G enhances cell survival and accelerates DNA repair processes, particularly the non-homologous end-joining (NHEJ) repair pathway. Notably, PA2G treatment reduces the frequency of lethal chromosomal aberrations and micronuclei formation, indicating its ability to enhance the repair of complex DNA lesions. Furthermore, PA2G is shown to play a role in potentially lethal damage repair (PLDR). These radioprotective effects are specific to NHEJ and ATM pathways, as cells deficient in these mechanisms do not benefit from PA2G treatment. This study highlights PA2G as a versatile radioprotector, both pre- and post-irradiation, with significant potential for applications in radiation therapy and protection, offering new insights into its mechanism of action. Further research is required to elucidate the precise molecular mechanisms underlying PA2G's radiomitigation effects and its potential clinical applications.

Biophysical Model Including a Potentially Lethal Damage Repair Parameter in Fractionated Carbon Beam

The amount of potentially lethal damage repair (PLDR) is a significant factor in the process of modeling the survival curves of cells irradiated with fractionated carbon beams. Because the amount of PLDR generally depends on the features of the cells and the linear energy transfer (LET), the amount of PLDR of cells irradiated with fractionated carbon beams shows distinct differences from that of cells irradiated with X-rays. This study considered a new parameter dependent on the correlation between the PLDR trait (T) of the cells over a time interval (Δ) at the fractionated carbon beam irradiation. The survival curves of the cells irradiated with fractionated carbon beams n times were predicted using the ζ and the Ψ values from the delay assay. This study aims to overcome the barriers of traditional methods by developing a new survival curve model with new parameters based on an analysis of the PLDR traits of cells over time interval in fractionated carbon beam irradiation and to suggest a model that produces results significantly closer to the experimental data.

Effect of X-ray minibeam radiation therapy on clonogenic survival of glioma cells

The goal is to compare, in vitro, the efficiency of minibeam radiotherapy (MBRT) and standard RT in inducing clonogenic cell death in glioma cell lines. With this aim, we report on the first in vitro study performed in an X-ray Small Animal Radiation Research Platform (SARRP) modified for minibeam irradiations. F98 rat and U87 human glioma cells were irradiated with either an array of minibeams (MB) or with conventional homogeneous beams (broad beam, BB). A specially designed multislit collimator was used to generate the minibeams with a width of a center-to-center distance of 1465 (±10) μm, and a PVDR value of 12.4 (±2.3) measured at 1 cm depth in a water phantom. Cells were either replated for clonogenic assay directly (immediate plating, IP) or 24 h after irradiation (delayed plating, DP) to assess the effect of potentially lethal damage repair (PLDR) on cell survival. Our hypothesis is that with MBRT, a similar level of clonogenic cell death can be reached compared to standard RT, when using equal mean radiation doses. To prove this, we performed dose escalations to determine the minimum integrated dose needed to reach a similar level of clonogenic cell death for both treatments. We show that this minimum dose can vary per cell line: in F98 cells a dose of 19 Gy was needed to obtain similar levels of clonogenic survival, whereas in U87 cells there was still a slightly increased survival with MB compared to BB 19 Gy treatment.The results suggest also an impairment of DNA damage repair in F98 cells as there is no difference in clonogenic cell survival between immediately and delayed plated cells for each dose and irradiation mode. For U87 cells, a small IP-DP effect was observed in the case of BB irradiation up to a dose of 17 Gy. However, at 19 Gy BB, as well as for the complete dose range of MB irradiation, U87 cells did not show a difference in clonogenic survival between IP and DP. We therefore speculate that MBRT might influence PLDR. The current results show that X-ray MBRT is a promising method for treatment of gliomas: future preclinical and clinical studies should aim at reaching a minimum radiation (valley) dose for effective eradication of gliomas with increased sparing of normal tissues compared to standard RT.

Radiosensitivity of quiescent and proliferating cells grown as multicellular tumor spheroids.

The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination‐based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia‐inducible factor‐1α‐positive but pimonidazole‐negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long‐term observation of cell kinetics inside of the spheroid following irradiation using real‐time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments.

Dynamics of chromosomal aberrations, induction of apoptosis, BRCA2 degradation and sensitization to radiation by hyperthermia.

Hyperthermia can transiently degrade BRCA2 and thereby inhibit the homologous recombination pathway. Induced DNA-double strand breaks(DSB) then have to be repaired via the error prone non-homologous end-joining pathway. In the present study, to investigate the role of hyperthermia in genotoxicity and radiosensitization, the induction of chromosomal aberrations was examined by premature chromosome condensation and fluorescence insitu hybridisation(PCC-FISH), and cell survival was determined by clonogenic assay shortly(0-1h) and 24h following exposure to hyperthermia in combination with ionizing radiation. Prior to exposure to 4Gy γ-irradiation, confluent cultures of SW‑1573(human lung carcinoma) and RKO(human colorectal carcinoma) cells were exposed to mild hyperthermia(1h, 41˚C). At 1h, the frequency of chromosomal translocations was higher following combined exposure than following exposure to irradiation alone. At 24h, the number of translocations following combined exposure was lower than following exposure to irradiation only, and was also lower than at 1h following combined exposure. These dynamics in translocation frequency can be explained by the hyperthermia-induced transient reduction of BRCA2 observed in both cell lines. In both cell lines exposed to radiation only, potentially lethal damage repair(PLDR) correlated with a decreased number of chromosomal fragments at 24h compared to 1h. With combined exposure, PLDR did not correlate with a decrease in fragments, as in the RKO cells at 24h following combined exposure, the frequency of fragments remained at the level found after 1h of exposure and was also significantly higher than that found following exposure to radiation alone. This was not observed in the SW‑1573 cells. Cell survival experiments demonstrated that exposure to hyperthermia radiosensitized the RKO cells, but not the SW‑1573 cells. This radiosensitization was at least partly due to the induction of apoptosis, which was only observed in the RKO cells and which may have been induced by BRCA2 degradation or different types of chromosomal aberrations. An important observation of this study is that the genotoxic effect of hyperthermia shortly after combined epxosure (to hyperthermia and radiation) is not observed at 24h after treatment.

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.

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD2). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a 137Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Decay of γ-H2AX foci correlates with potentially lethal damage repair in prostate cancer cells

To determine the relationship between ionizing radiation-induced levels of γ-H2AX foci and cell survival in cultured prostate cancer cell lines, three prostate cancer cell lines: LNCaP (wt TP53), DU145 (mut TP53) and PC3 (TP53 null), were studied. For γ-H2AX foci induction, cells were irradiated with a single dose of 2 Gy and foci levels were studied at 30 min and 24 h after irradiation. Cell survival was determined by clonogenic assay, directly and 24 h after irradiation with doses ranging from 0 to 8 Gy. Irradiation was performed with a Siemens Stabilipan 250 KeV X-ray machine at a dose rate of approximately 3 Gy/min. Survival curves were analyzed using the linear-quadratic model S(D)/S(0)=exp-(αD+βD2). LNCaP cells clearly demonstrated potentially lethal damage repair (PLDR) which was assessed as increased survival levels after delayed plating as compared to cells plated immediately after irradiation. DU145 cells demonstrated only a slight PLDR and PC3 cells did not show PLDR at all. Levels of γ-H2AX foci were significantly decreased in all cell lines at 24 h after irradiation, compared to levels after 30 min. The LNCaP cells which demonstrated a clear PLDR also showed the largest decay in the number of γ-H2AX foci. In addition, the PC cells which did not show PLDR had the lowest decay of γ-H2AX foci. A clear correlation was demonstrated between the degree of decay of γ-H2AX foci and PLDR.

Modeling the biological response of normal human cells, including repair processes, to fractionated carbon beam irradiation

To understand the biological response of normal cells to fractionated carbon beam irradiation, the effects of potentially lethal damage repair (PLDR) and sublethal damage repair (SLDR) were both taken into account in a linear-quadratic (LQ) model. The model was verified by the results of a fractionated cell survival experiment with normal human fibroblast cells. Cells were irradiated with 200-kV X-rays and monoenergetic carbon ion beams (290 MeV/u) at two irradiation depths, corresponding to linear energy transfers (LETs) of approximately 13 keV/μm and 75 keV/μm, respectively, at the Heavy Ion Medical Accelerator in Chiba of the National Institute of Radiological Sciences. When we only took into account the repair factor of PLDR, γ, which was derived from the delayed assay, the cell survival response to fractionated carbon ion irradiation was not fully explained in some cases. When both the effects of SLDR and PLDR were taken into account in the LQ model, the cell survival response was well reproduced. The model analysis suggested that PLDR occurs in any type of radiation. The γ factors ranged from 0.36–0.93. In addition, SLD was perfectly repaired during the fraction interval for the lower LET irradiations but remained at about 30% for the high-LET irradiation.

Intercellular Communication Amplifies Stressful Effects in High-Charge, High-Energy (HZE) Particle-Irradiated Human Cells

Gap junction intercellular communication/Cell killing/Potentially lethal damage repair/DNA damage/ Linear energy transfer of space radiation. Understanding the mechanisms that underlay the biological effects of particulate radiations is essential for space exploration and for radiotherap y. Here, we investigated the role of gap junction intercellular communication (GJIC) in modulating harmful effects induced in confluent cultures wherein most cells are traversed by one or more radiation tracks. We focused on the effect of radiation quality (linear energy transfer; LET) on junctional propagation of DNA damage and cell death among the irradiated cells. Confluent normal human fibroblasts were exposed to gr aded doses of 1 GeV protons (LET ~0.2 keV/μm) or 1 GeV/u iron ions (LET ~151 keV/μm) and were assayed for clonogenic survival and for micronucleus formation, a reflection of DNA damage, shortly after irradiation and following longer incubation periods. Iron ions were ~2.7 fold more effective than protons at killing 90% of the cells in the exposed cultures when assayed within 5–10 minutes after irradiation. When cells were held in the confluent state for several hours after irradiation, substantial potentially lethal damage repair (PLDR), coupled with a reduction in micronucleus formation, occurred in cells exposed to protons, but not in those exposed to iron ions. In fact, such confluent holding after exposure to a similarly toxic dose of iron ions enhanced the induced toxic effect. However, following iron ion irradiation, inhibition of GJIC by 18-α-glycyrrhetinic acid eliminated the enhanced toxicity and red uced micronucleus formation to levels below those detected in cells assayed shortly after irradiation. The data show that low-LET radiation induces strong PLDR within hours, but that high-LET radiation with similar immediate toxicity does not induce PLDR and its toxicity increases with time following irradiation. The results also show that GJIC among irradiated cells amplifies stressful effects following exposure to high-, but not low-LET radiation, and that GJIC has only minimal effect on cellular recovery following low-LET irradiation.

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.

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.

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Radiosensitivity of hypoxic and proliferating clonogen in a human lung cancer grown in nude mice

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we studied their radiosensitivity by focusing attention on the dynamics of tumor clonogens. The movement of clonogens in the regrowing IA tumor after irradiation can be divided into three phases: first, the early and rapid survival recovery (PLD repair) phase; second, the delay phase involving a certain lag in survival change; and third, the repopulation phase consisting of two stages: the anoxic repopulation before angiogenesis and the hypoxic repopulation in the presence of a poorly developed vascular network. Clonogens in a regrowing tumor after irradiation were found to actively proliferate even in an anoxic environment before angiogenesis and under the hypoxic conditions prevailing after the formation of a tumor with a poorly developed vascular system. This re-grown tumor was found to be more radioresistant than a sham-treated control tumor. It is believed that these clonogens are genetically selected under hypoxic conditions throughout the process of tumor growth and regrowth, and may be primarily involved in tumor recurrence or accelerated repopulation in fractionated irradiation.

The role of Raf-1 in radiation resistance of human hepatocellular carcinoma Hep G2 cells

Development of radiation resistance is one of the major reasons that cancer cells do not respond to radiotherapy and the mechanism for resistance is still not clear. Two sublines of human hepatocellular carcinoma Hep G2 cells were established from cells that survived two different irradiation regimes, 2 Gy for 10 days or 10 Gy for 2 days, respectively. Using MTT assay, the radiation conditioned cells were found to be more resistant to gamma-irradiation and have a greater extent of potentially lethal damage repair (PLDR) for radiation than the parent cells. By Western blot analysis, the radiation-conditioned cells were found to overexpress Raf-1 which is known to regulate the radiation resistance of cells. Inhibition of Raf-1 expression by antisense oligonucleotides increased the radiation sensitivity of the radiation-conditioned cells while inhibitors of Ras (L744,832), PI3K (LY294002) and p38 (SB203580) had no effect. Moreover, antisense Raf-1 oligonucleotides also decreased the radiation induced PLDR capacity of the radiation conditioned cells. It is therefore suggested that Raf-1 may induce radiation resistance through an increase in radiation induced PLDR capacity in Hep G2 cells.

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.

Induction of the early response protein EGR-1 in human tumour cells after ionizing radiation is correlated with a reduction of repair of lethal lesions and an increase of repair of sublethal lesions

The role of EGR-1 in potentially lethal damage repair (PLDR) was studied. Induction of the early response protein EGR-1 and survival after ionizing radiation of two human tumour cell lines after culturing for 48 h in serum-deprived medium was investigated. The glioblastoma cell line (Gli-6) and a lung carcinoma cell line (SW-1573) were selected as these cell lines differ considerably in the degree of PLD repair after radiation. In both cell lines induction of EGR-1 protein was observed between 30-120 min after treatment with 10 Gy in serum-deprived cultures. In cells growing in medium with normal serum no induction of EGR-1 was observed. No difference in EGR-1 expression levels between the two cell lines was detected. Linear-Quadratic analysis of the survival curves showed a much larger difference between the values of alpha after immediate and delayed plated cells of the cultures in normal serum as compared to cells cultured in serum-deprived medium. The cells cultured in serum-deprived medium showed much larger difference between the values of beta. This indicates that induction of EGR-1 is correlated with a reduction of repair of lethal lesions (PLDRalpha) and with an increase of repair of sublethal lesions (PLDRbeta).

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.