The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5–8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G 1 cells to 137 Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.

We previously described an enhanced sensitivity for cell killing and γ-H2AX focus induction after both high-dose-rate and continuous low-dose-rate γ irradiation in 14 primary fibroblast strains derived from hereditary-type retinoblastoma family members (both affected RB1 +/− probands and unaffected RB1 +/+ parents). Here we present G 2 -phase chromosomal radiosensitivity assay data for primary fibroblasts derived from these RB family members and five Coriell cell bank controls (four apparently normal individuals and one bilateral RB patient). The RB family members and two normal Coriell strains had significantly higher (∼1.5-fold, P < 0.05) chromatid-type aberration frequencies in the first postirradiation mitosis after doses of 50 cGy and 1 Gy of 137 Cs γ radiation compared to the remaining Coriell strains. The induction of chromatid-type aberrations by high-dose-rate G 2 -phase γ irradiation is significantly correlated to the proliferative ability of these cells exposed to continuous low-dose-rate γ irradiation (reported in Wilson et al. , Radiat. Res. 169, 483–494, 2008). Our results suggest that these moderately radiosensitive individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes or may carry epigenetic changes involving genes that more broadly modulate such systems, including G 2 -phase-specific DNA damage responses.

In addition to cell cycle arrest, DNA repair or/and apoptosis, ionizing radiation can also induce premature senescence, which could lead to very different biological consequences depending on the cell type. We show in this report that low-dose radiation-induced senescent stromal fibroblasts stimulate proliferation of cocultured breast carcinoma cells. Such effects of senescent fibroblasts appear to result from their ability to induce the expression in carcinoma cells of mitotic genes and subsequent mitotic division. The elevated proliferation of breast carcinoma cells correlates with resistance to radiation as well as to adriamycin. Of interest is the observation that exposure to lower doses (<20 cGy) augments the ability of senescent fibroblasts to promote the survival of cocultured breast carcinoma cells. The resistance appears to be mediated partially by the Akt pathway, because expression of a dominant negative Akt mutant in breast carcinoma cells results in a partial reversal of the radioresistance. The ability of fibroblasts to modulate the radiosensitivity of nearby carcinoma cells implicates the importance of targeting the stroma during therapy.

Wilson, P. F., Nagasawa, H., Warner, C. L., Fitzek, M. M., Little, J. B. and Bedford, J. S. Radiation Sensitivity of Primary Fibroblasts from Hereditary Retinoblastoma Family Members and Some Apparently Normal Controls: Colony Formation Ability during Continuous Low-Dose-Rate Gamma Irradiation. Radiat. Res. 169, 483–494 (2008). We previously described an enhanced sensitivity for cell killing and G 1 -phase cell cycle arrest after acute γ irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1 +/− probands and unaffected RB1 +/+ parents) as well as distinctive gene expression profiles in unirradiated cultures by microarray analyses. In the present study, we measured the colony formation ability of these cells after exposure to continuous low-dose-rate (0.5–8.4 cGy/h) 137 Cs γ radiation for a 2-week growth period. Fibroblasts from all RB family members (irrespective of RB1 genotype) and from 5 of 18 apparently normal Coriell cell bank controls were significantly more radiosensitive than the remaining apparently normal controls. The average dose rates required to reduce relative survival to 10% and 1% were ∼3.1 and 4.7 cGy/h for the Coriell control strains with normal radiosensitivity and ∼1.4 and 2.5 cGy/h for the radiosensitive RB family member and remaining apparently normal Coriell control strains. The finding that a significant proportion of fibroblast strains derived from apparently normal individuals are sensitive to chronic low-dose-rate irradiation indicates such individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes that may likewise predispose them or their children to cancer.

Little, J. B., Nagasawa, H., Li, G. C. and Chen, D. J. Involvement of the Nonhomologous End Joining DNA Repair Pathway in the Bystander Effect for Chromosomal Aberrations. Radiat. Res. 159, 262–267 (2003). Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G 1 phase and exposed to very low fluences of α particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4–9% of wild-type cells and 36–55% of Xrcc5 −/− cells, whereas only 2–3% of the nuclei were traversed by an α particle and thus received any radiation exposure. G22p1 −/− cells responded similarly to Xrcc5 −/− cells, whereas Prkdc −/− and Adprt −/− cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5 −/− and G22p1 −/− cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc −/− cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

Schäfer, J., Bachtler, J., Engling, A., Little, J. B., Weber, K.-J. and Wenz, F. Suppression of Apoptosis and Clonogenic Survival in Irradiated Human Lymphoblasts with Different TP53 Status. Radiat. Res. 158, 699–706 (2002). The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G 2 /M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.

Little, J. B., Nagasawa, H., Dahlberg, W. K., Zdzienicka, M. Z., Burma, S. and Chen, D. J. Differing Responses of Nijmegen Breakage Syndrome and Ataxia Telangiectasia Cells to Ionizing Radiation. Radiat. Res. 158, 319–326 (2002). Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G 0 /G 1 phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G 1 -phase arrest. Irradiated AT cells showed no potentially lethal damage and no G 1 -phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21 Waf1 ) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.

Huo, L., Nagasawa, H. and Little, J. B. HPRT Mutants Induced in Bystander Cells by Very Low Fluences of Alpha Particles Result Primarily from Point Mutations. Radiat. Res. 156, 521–525 (2001). We have shown previously that damage signals may be transmitted from irradiated cells to nonirradiated cells in monolayer cultures, leading to changes in gene expression and an enhanced frequency of mutations in these “bystander” cells. The present study was designed to test the hypothesis that mutations occurring in bystander cells result from a different mechanism than those occurring in irradiated cells, and thus show differences in molecular structure. Structural changes in the HPRT gene of Chinese hamster ovary (CHO) cells were determined by multiplex PCR analysis. A total of 790 mutant clones derived from monolayer cultures exposed to mean doses of 0, 0.5 or 10 cGy of α-particle radiation (0, 3% or 44%, respectively, of nuclei traversed by one or more α particles) were examined. Whereas mutations induced by 10 cGy included a high frequency of deletions, nearly all mutations occurring in bystander cells in cultures irradiated with 0.5 cGy involved point mutations, confirming our hypothesis that they are induced by a different mechanism.

Syljuåsen, R. G., Krolewski, B. and Little, J. B. Molecular Events in Radiation Transformation. Studies of human tumor cell lines have revealed alterations in the regulation of a number of cell cycle-related genes, associated in some cases with a TP53 -independent loss of the radiation-induced G 1 -phase arrest. It is not clear, however, whether these are early or late events in tumor development, or they arise in tumor cell lines during growth in culture. Since the oncogenic transformation of an individual cell is thought to be an early event in tumor development, we have used a model system of normal and radiation-transformed C3H 10T½ mouse fibroblast cell clones to address this issue. Transformed clones derived from type III foci were compared with clones derived from parental, wild-type cells. Approximately 25% of transformed clones showed Trp53 mutations in exon 5; however, preliminary results based on in situ immunofluorescence studies with an antibody recognizing mutant Trp53 indicate that the appearance of such mutations in transformed clones occurs late in the process of transformation and is unlikely to represent an initiating event. The remaining transformed clones and all clones derived from parental cells expressed wild-type Trp53. Radiation-induced G 1 -phase arrest was either absent or significantly reduced in all of the transformed clones, independent of Trp53 status. Constitutive expression of Cdkn1a protein was significantly increased in most of the transformed clones. Also, the majority of transformed clones showed elevated levels of cyclin D1, and two clones overexpressed cyclin E. These results indicate that loss of G 1 -phase checkpoint control, independent of Trp53 status, and altered expression of cell cycle regulatory proteins may represent early events in the process of radiation-induced carcinogenesis that are associated with the malignant transformation of individual cells.

We examined the induction of HPRT mutations in CHO cells exposed to low fluences of <tex-math>${}^{238}{\rm Pu}$</tex-math> α particles from a specially constructed irradiator. The dose-response relationship was linear over the dose range of 5 cGy-1.2 Gy. However, unexpected sensitivity, leading to a significantly higher frequency of mutations than would be predicted by a back extrapolation from the data for higher doses, was observed in the dose range below 5 cGy, where the mean number of α-particle traversals per nucleus was significantly less than one (0.05-0.3). The frequency of mutations induced by a single α particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied. The data are consistent with the conclusion that the enhanced efficiency of each nuclear traversal at low particle fluences is the result of mutations arising in nonirradiated, bystander cells.

We demonstrate by western analysis that the expression levels of TP53 (formerly known as p53), CDKN1A (formerly known as ${\rm p}21^{{\rm Waf1}}$ ), CDC2 (formerly known as ${\rm p}34^{{\rm cdc2}}$ ), CCNB1 (cyclin B1) and RAD51 are significantly modulated in confluent, density-inhibited human diploid cell populations exposed to doses where only a small fraction of the nuclei are actually traversed by an α-particle track. The extent of modulation of TP53 and CDKN1A is significantly reduced in the presence of the gap junction inhibitor lindane and in irradiated low-density cell populations. In situ immunofluorescence studies show that at doses where about 2% of the nuclei would be traversed by an α particle, induction of CDKN1A occurs in more cells than predicted. Furthermore, the induced cells are present in isolated aggregates of neighboring cells. Therefore, our studies at the gene expression level indicate that similar signaling pathways are induced in bystander cells that are not traversed by an α particle as in traversed cells, and that biological effects in cell populations are not restricted to the response of individual cells to the DNA damage they receive.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase δ and ε involved in DNA replication and nucleotide excision repair. There are two intranuclear fractions: a detergent-extractable, soluble fraction and a tightly DNA-bound fraction. To function, PCNA forms a trimeric sliding clamp which is loaded onto DNA. To better understand the role of the p53/p21 pathway in the regulation of PCNA after irradiation, we studied three closely related human lymphoblastoid cell lines, WTK1, TK6 and TK6E6, an HPV16 E6-transfected line, that differ in p53 status, radiosensitivity and susceptibility to radiation-induced apoptosis. Time-dependent changes in PCNA levels were measured in the different nuclear fractions by Western blot analysis after protein crosslinking. The results were compared to those for human diploid fibroblasts studied under different growth conditions. There was no change in total cellular levels of PCNA after irradiation, consistent with predominantly post-translational regulation. Changes in intranuclear distribution and complex formation occurred in a p53/p21-dependent manner. The loading of PCNA onto DNA was increased in cells with low p21 levels. A disruption of PCNA trimers was observed in exponentially growing p53 + cells in the soluble fraction. Thus the p53/p21 signal transduction pathway appears to play a significant role in the regulation of the response of PCNA to radiation.

The frequency of mutations at the Hprt locus was measured in clonal populations of Chinese hamster ovary cells derived from single cells surviving exposure to 0-12 Gy of X rays or 2 Gy of α particles. Approximately 8-9% of 446 clonal populations examined 23 population doublings after irradiation showed high frequencies of late-arising mutations as indicated by mutant fractions $10^{2}-10^{4}\text{-fold}$ above background. The frequency with which such clones occurred was similar for α-particle irradiation and X irradiation, with no apparent dose dependence for X irradiation over the range of 4-12 Gy. The molecular structure of Hprt mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. Of mutations induced directly after exposure to X rays, 75% involved partial or total gene deletions. Only 19-23% of late-arising (delayed) mutations were associated with deletions, the preponderance of these being partial deletions involving one or two exons. This spectrum was very similar to that for spontaneously arising mutations. To determine whether delayed mutations were non-clonal, the spectrum of exons deleted was examined among 29 mutants with partial deletions derived from a single clonal population. The results indicated that at least 15 of these mutants arose independently. To examine the relationship between the occurrence of delayed mutations and chromosomal instability, 60 Hprt mutant subclones isolated from a clonal population showing a high frequency of delayed mutations were serially cultivated in vitro. Of these, 14 showed a slow-growth phenotype with a high frequency of polyploid cells (10-38%) and a markedly enhanced frequency of non-clonal chromosomal rearrangements including both chromosome-type and chromatid-type aberrations. These clones also showed a 3- to 30-fold increase in the frequency of ouabain-resistant mutations; no ouabain-resistant mutants were induced directly by X irradiation. These results suggest that among clones showing a high frequency of delayed mutations there may be a subpopulation of cells that are particularly unstable; selection for the slow-growth phenotype has the effect of selecting for this chromosomally unstable subpopulation.

It has been shown that p53 - human colorectal cancer cells arrest after DNA damage in a ${\rm G}_{2}\text{-like}$ state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the ${\rm G}_{2}/{\rm M}\text{-phase}$ checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy γ rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53 - cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53 - WTK1 line was due to the occurrence of a G 2 -phase block in a pre-existing tetraploid population. These results support the conclusion that control of the ${\rm G}_{2}/{\rm M}\text{-phase}$ checkpoint after irradiation may differ among different cell types.

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.