Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells.

PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. CONCLUSIONS: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

G6PD deficient cells and the bioreduction of disulfides: effects of DHEA, GSH depletion and phenylarsine oxide.

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation.

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line

Involvement of energy metabolism in the production of 'bystander effects' by radiation.

These experiments were done to determine if interference with energy metabolism and REDOX biochemistry during low LET radiation exposure would alter the ability of medium harvested from the irradiated cells to induce a bystander effect in unirradiated cells. Human keratinocyte cells and CHO-K1 mutant cell lines were irradiated using cobalt 60. Clonogenic assays were used to determine the reproductive death of the cells exposed to direct irradiation or medium from irradiated cells. The persistence in progeny was also examined. Use of apoptosis inhibitors or medium from the LDH or G6PD null cell lines, reduced or prevented the bystander effect. Transfection with G6PD recovered the effect. Treatment with anti-oxidant substances, L-lactate and L-deprenyl prevented bystander factor associated cell kill. The lactate analogue, oxamate, was less effective. Data from experiments where media harvested from the different cell lines was exchanged suggest that signal production and cellular response may involve different mechanisms. The effects on exposed cells were transmitted to progeny which also showed excessive levels of cell death for several generations. The results suggest that energy/REDOX metabolism may be involved in the expression of a radiation induced bystander response. Given the aberrant energy metabolism in tumour cells, this may have implications for dose escalation in radiotherapy.

Radiation-induced recombination is dependent on Ku80.

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis.

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Absence of DNA ligase IV protein in XR-1 cells: evidence for stabilization by XRCC4.

XR-1 is a CHO mutant cell line defective in double strand break repair and V(D)J recombination. These defects are due to a deletion of the XRCC4 gene which encodes a 38-kDa nuclear phosphoprotein. Recent studies have shown that XRCC4 interacts with and enhances the activity of DNA ligase IV in vitro. In this study we investigate the effect of the absence of XRCC4 on the level of DNA ligase IV in XR-1 cells. Western blot analysis indicates that levels of DNA ligase IV protein are almost undetectable in these cells, however, introduction of the XRCC4 cDNA into XR-1 resulted in a return to wild type levels of the protein. Furthermore, analysis of DNA ligase IV mRNA showed equivalent levels in both XR-1 and XRCC4 transfected XR-1 indicating that the altered level of DNA ligase IV is not due to a change in the expression of the gene. These data strongly suggest that an important function of XRCC4 is to stabilize the DNA ligase IV protein.

Time versus replication dependence of EMS-induced delayed mutation in Chinese hamster cells.

We have previously observed in Chinese hamster cells that ethyl methane sulfonate (EMS) induces mutations which are distributed over at least 10-14 cell divisions following treatment. This delayed appearance of mutations could be explained by EMS-induced lesions which remain in DNA and have a probability that is significantly less than 1.0 of producing base mispairing errors during successive replication cycles (replication-dependent). Alternatively, delayed mutation may be a time-dependent process in which a slow acting or damage inducible error-prone repair process removes persistent DNA lesions and replaces them with an incorrect base during the course of 7-10 days of colony growth following EMS exposure. To address this question, the distribution of HGPRT delayed mutation events (fifth division or later) in cells plated immediately for exponential growth after EMS treatment was compared with the distribution in cells which remained under confluent growth conditions for 8 days and then were replated. Both the distribution and rate of accumulation of delayed mutations (mutations/cell division) were similar in the two culture conditions. In contrast, the frequency of early mutations (before the fifth division) in the confluent population was reduced more than 2-fold compared to dividing cells. A comparison of the frequency of EMS-induced DNA lesions in the two populations revealed that the density inhibited population contained one third the DNA lesions of the exponential population. These results argue against a time-dependent process since, if this mechanism applies, one would expect an increase in early mutant events and a decrease in delayed events in the confluent population. The results, however, are consistent with a replication model in which potential early mutant lesions are preferentially removed in the density inhibited culture during the 8 days of incubation while lesions producing late mutants are not removed.

Double-strand break repair in Ku86- and XRCC4-deficient cells.

The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells.

V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed.

The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase.

Solid tumours develop an acidic extracellular environment with high concentration of lactic acid, and lactic acid produced by glycolysis has been assumed to be the major cause of tumour acidity. Experiments using lactate dehydrogenase (LDH)-deficient ras-transfected Chinese hamster ovarian cells have been undertaken to address directly the hypothesis that lactic acid production is responsible for tumour acidification. The variant cells produce negligible quantities of lactic acid and consume minimal amounts of glucose compared with parental cells. Lactate-producing parental cells acidified lightly-buffered medium but variant cells did not. Tumours derived from parental and variant cells implanted into nude mice were found to have mean values of extracellular pH (pHe) of 7.03 +/- 0.03 and 7.03 +/- 0.05, respectively, both of which were significantly lower than that of normal muscle (pHe = 7.43 +/- 0.03; P < 0.001). Lactic acid concentration in variant tumours (450 +/- 90 microg g(-1) wet weight) was much lower than that in parental tumours (1880 +/- 140 microg/g(-1)) and similar to that in serum (400 +/- 35 microg/g(-1)). These data show discordance between mean levels of pHe and lactate content in tumours; the results support those of Newell et al (1993) and suggest that the production of lactic acid via glycolysis causes acidification of culture medium, but is not the only mechanism, and is probably not the major mechanism responsible for the development of an acidic environment within solid tumours.

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: involvement of the V(D)J recombination double-strand break repair pathway.

Repair of DNA double-strand breaks (DSBs) is linked to the V(D)J recombination pathway through investigations of radiation-sensitive mutants. Here we report a possible association between the distribution of DSBs within higher-order chromatin structures and this pathway. Both murine severe combined immunodeficient (SCID) and Chinese hamster XR-1 cells exhibit defective DNA DSB repair and defective V(D)J recombination. The DSB repair defect is not complete, with only a subset of slowly repairing lesions affected by the mutations in these cell lines. We used a modified neutral filter elution procedure which retained elements of higher-order chromatin structures, namely nuclear matrix-DNA interactions. X-ray-induced DSBs that occurred as multiples within looped DNA structures were nonrepairable in SCID and XR-1 cells. In contrast, these lesions were repaired in radioresistant wild-type cells. Cell lines complemented with human DNA containing the respective complementing genes (XRCC7 and XRCC4) showed an increased rate of DSB repair. These results agree with previous findings with xrs5 cells (a member of the XRCC5 group). Xrs5 cells are defective for the Ku p80 subunit of the V(D)J recombination complex and show repair and V(D)J recombination defects similar to those of SCID and XR-1 cells.

EMS and UV-light-induced colony sectoring and delayed mutation in Chinese hamster cells.

PURPOSE: To review studies of mutagen-induced colony sectoring which demonstrate that UV light and EMS produce delayed mutational events in Chinese hamster ovary cells. METHODS AND RESULTS: Since the late 1940s, it has been known that the treatment of a single bacterial or yeast cell with mutagenic agents produces complete mutant colonies (pures) and colonies composed of both mutant and non-mutant cell types (mosaics) with various sectored patterns. A similar sectoring phenomenon has been observed in Chinese hamster ovary cells (CHO) using the DNA alkylating agent ethyl methane sulphonate (EMS) or ultraviolet light. However, unlike bacteria and yeast, a significant fraction of CHO mutant colonies contained sectors of less than 1/2; i.e. 1/4, 1/8 and 1/16 sectors, suggesting a delayed production of mutations. Using various colony-replating approaches, it was found that these mutagenic agents produced the ratio of mutant to wild-type cells expected for a delayed mutational process which produces mutant events for at least 12-14 cell divisions following treatment. This delayed mutation phenomenon was observed at both the glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) loci. Various mutational mechanisms for the production of delayed mutations are discussed. CONCLUSIONS: These studies suggest that mutagens such as UV light and EMS induce long-term alterations in mammalian cells that act to increase the 'apparent' spontaneous mutation frequency. This delayed mutational decrease in stability of the genome may explain the accumulation over time of the multiple genetic changes observed in malignant tumours.

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination.

The XR-1 Chinese hamster ovary cell line is impaired in DNA double-strand break repair (DSBR) and in ability to support V(D)J recombination of transiently introduced substrates. We now show that XR-1 cells support recombination-activating gene 1- and 2-mediated initiation of V(D)J recombination within a chromosomally integrated substrate, but are highly impaired in ability to complete the process by forming coding and recognition sequence joins. On this basis, we isolated a human cDNA sequence, termed XRCC4, whose expression confers normal V(D)J recombination ability and significant restoration of DSBR activity to XR-1, clearly demonstrating that this gene product is involved in both processes. The XRCC4 gene maps to the previously identified locus on human chromosome 5, is deleted in XR-1 cells, and encodes a ubiquitously expressed product unrelated to any described protein.

Measurement of right ventricular volume by conductance catheter in closed-chest pigs.

This study details the effects of changes in right ventricular (RV) volume on the conductance catheter gain factor both over a broad volume range and within the cardiac cycle. In seven closed-chest anesthetized pigs, a conductance catheter was introduced transvenously and positioned to span the RV long axis, including the outflow tract. Parallel conductance was determined using a saline dilution technique. Conductance volume gain factor (alpha) was computed using stroke volume obtained by thermodilution over a range of volumes obtained by volume loading or sustained partial occlusion of the inferior caval vein. The chest was then opened, an ultrasonic flow probe was placed around the pulmonary artery, and the conductance-derived RV volume was compared with the pulmonary flow integral over the course of ejection. When volume was varied over a broad range, an inverse relation between RV volume and alpha was observed (P < 0.001). This did not cause significant nonlinearity of the conductance-volume relation. The relation was also relatively linear during the course of ejection within the cardiac cycle. These results indicate that the conductance catheter may be employed, using the described technique, to assess RV volume under steady-state conditions.

Transcatheter closure of an aortopulmonary window with a modified double umbrella occluder system.

Transcatheter occlusion of a small aortopulmonary window was successfully performed in a child using a double umbrella occluding device. The delivery system was adapted for use through a small sheath from a transvenous approach. Transcatheter closure is feasible in appropriately selected aortopulmonary windows.

Regional localization of the XRCC4 human radiation repair gene.

To map the XRCC4 human DNA repair gene subchromosomally, a gamma-ray-resistant human:XR-1 hamster hybrid cell containing fragments of human chromosome 5 and the pSV2neo plasmid was lethally irradiated and fused with the gamma-ray-sensitive XR-1 mutant cell. After selection for G418 resistance, 2 of a total of 76 hybrids retained wildtype gamma-ray resistance. FISH analysis of normal human lymphocytes using DNA from the two resistant hybrids as probes produced a common region of hybridization at 5q13-q14, suggesting that the XRCC4 gene is in this region of chromosome 5. The gene was physically localized between D5S427 and D5S401 microsatellite markers and the cytological assignment confirmed using hamster:human hybrids containing known deletions in human chromosome 5.

UV-light induces delayed mutations in Chinese hamster cells.

The possibility was examined that mutational events at the glucose 6-phosphate dehydrogenase locus can be delayed for significantly more than one or two cell divisions following treatment of Chinese hamster cells with UV light. To detect these later mutant events, the proportion of G6PD-mutant cells in a colony was obtained by replating cells from a single colony 5-7 days after UV irradiation and staining the resulting colonies for G6PD activity. Eight colonies out of a total of 1657 colonies from the treated population yielded G6PD-negative colonies upon replating, while no mutant clones were obtained from 947 colonies grown from untreated cells. One clone contained only mutant cells suggesting that the mutation occurred before the first cell division after irradiation. A second clone contained a 1:1 ratio of mutant to wild-type cells suggesting that the mutation occurred at the first cell division. The remaining six clones contained low numbers of mutant cells and the ratio of mutant to wild-type cells in these clones was in agreement with the theoretical ratios expected for mutations occurring at the fourth (1:13), sixth (1:63), seventh (1:127), eighth (1:255), ninth (1:511), and eleventh (1:2047) cell division after UV light exposure. G6PD-mutant cells deficient in staining activity were isolated from five clones and all have significant reductions in G6PD activity confirming the genetically stable character of these mutants. These results support the conclusion that UV light induces mutants for up to 11 cell generations after treatment of mammalian cells and suggest that mutagens can induce in mammalian DNA long-term alterations which act to increase the apparently spontaneous mutation frequency.