Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.     

Complementation of a DNA repair deficiency in six human tumor cell lines by chromosome 11

Summary Human tumor cells, after x-irradiation during the G₂ phase of the cell cycle, show an abnormally high frequency of persistent chromatid breaks and gaps resulting from deficient DNA repair. Addition of a single human chromosome 11 from normal fibroblasts by micro-cell fusion to cell lines from six different tumors resulted in efficient repair of the radiation-induced damage to the level in normal cells. For one of the cell lines, addition of the long arm of chromosome 11 was sufficient to restore repair efficiency. In four of the six tumor lines, restoration of efficient DNA repair by chromosome 11 was associated with tumor suppression in nude mice. These results suggest that chromosome 11 carries a DNA repair gene or genes that complement the repair deficiency of tumor cells and that this gene for at least one tumor is localized to the long arm.     

Normal DNA ligase activity in a gamma-ray-sensitive Chinese hamster mutant

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by gamma-irradiation during the sensitive G1--early-S period and somewhat deficient in repair of gamma-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5'-phosphate-3'-hydroxyl breaks in double-stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G1-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks

Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium. While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain. In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.     

Normal DNA ligase activity in a γ-ray-sensitive Chinese hamster mutant

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by γ-irradiation during the sensitive G₁-early-S period and somewhat deficient in repair of γ-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5′-phosphate-3′-hydroxyl breaks in double stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G₁-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.     

Genome Instability in Ataxia Telangiectasia (A-T) Families: Camptothecin-Induced Damage to Replicating DNA Discriminates between Obligate A-T Heterozygotes,A-T Homozygotes and Controls

Previously we used the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells primarily by inducing double strand breaks (DSBs) in replication forks, to show that ataxia telangiectasia (A-T) fibroblasts are defective in the repair of this particular subclass of DSBs. CPT treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges, viewed as prematurely condensed G2 chromosomes (G2 PCC), compared with normal cells where aberrations are mostly chromatid breaks. Here we show that A-T lymphoblastoid cells established from individuals with different mutations in the ATM gene also exhibit increased levels of chromosomal exchanges in response to CPT, indicating that the replication-associated DSBs are misrepaired in all these cells. From family studies we show that the presence of a single mutated allele in obligate A-T heterozygotes leads to intermediate levels of chromosomal exchanges in CPT-treated lymphoblastoid cells, thus providing a functional and sensitive assay to identify these individuals.     

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.     

DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange.

The Chinese hamster cell line mutant EM9, which has a reduced ability to repair DNA strand breaks, is noted for its highly elevated frequency of sister chromatid exchange, a property shared with cells from individuals with Bloom's syndrome. The defect in EM9 cells was corrected by fusion hybridization with normal human fibroblasts and by transfection with DNA from hybrid cells. The transformants showed normalization of sister chromatid exchange frequency but incomplete correction of the repair defect in terms of chromosomal aberrations produced by 5-bromo-2'-deoxyuridine.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells

In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G2, S and G1 phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G1 irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G1 irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G1 in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.     

Deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins in hyper-radiosensitive Long-Evans Cinnamon (LEC) rat cells after X irradiation

The DNA-dependent protein kinase (DNA-PK) complex has been implicated in the repair of DNA double-strand breaks (DSBs). DNA-PK is a heterotrimeric protein complex comprised of two components: a large catalytic subunit, Prkdc, with serine/threonine kinase activity and a DNA-targeting component, G22p1 and Xrcc5. In previous report, we showed that approximately 80% of the G22p1 and Xrcc5 proteins were observed in the cytoplasm of rat fibroblasts, and that nuclear translocation of the proteins from the cytoplasm is important for the repair of DNA DSBs. In the present study, we showed that nuclear accumulation of the G22p1 and Xrcc5 proteins was not observed in fibroblasts from a mutant strain of Long-Evans Cinnamon (LEC) rat that has an enhanced radiosensitivity and a reduced level of repair of DSBs after X irradiation. Nuclear translocation of the proteins was observed in both LEC rat cells and control rat cells with normal radiosensitivity at 5 min after X irradiation. Although high levels of G22p1 and Xrcc5 proteins were observed in the nuclei of control rat cells until 60 min postirradiation, the amounts of the proteins decreased rapidly in the nuclei of LEC rat cells in the first 10 min after X irradiation. These findings suggest that there are some defects in maintaining the levels of G22p1 and Xrcc5 proteins in the nuclei of LEC rat cells. An analysis of fibroblasts from backcross rats showed that the deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins is genetically linked to enhanced radiosensitivity. Since the nucleotide sequences of the G22p1 and Xrcc5 genes of the LEC rats coincided with those of the control rats, the deficiency in nuclear accumulation may not be caused by mutations of the G22p1 and Xrcc5 proteins.     

Characterization of a new radiation-sensitive mutant, Escherichia coli K-12 radC102

A new radiation-sensitive mutant, radC , has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for gamma-radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amounts of X- and uv-radiation-induced DNA degradation, and it was approximately 60% deficient in recombination ability. The radC strain was normal for host cell reactivation of gamma-and uv-irradiated bacteriophage lambda; the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, we suggest that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

Induction of chromatid breaks by carbon K-shell ultrasoft X rays

Chromatid breaks have previously been shown to be induced in G2-phase cells after exposure to ionizing radiation (X and gamma rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for 60Co gamma rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.