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PubMed1997

The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair.

Chinese hamster lung fibroblast V79 cells have been widely used in studies of DNA damage and DNA repair. Since the p53 gene is involved in normal responses to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79 cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was 76 and 75% homologous to human and mouse p53 respectively, and was 95% homologous to the Syrian hamster cells. The V79 p53 sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and nonfunctional. Our results indicate that caution should be used in interpreting studies of DNA damage, DNA repair and apoptosis in V79 cells.

Animals↗

PubMed2002

Interaction of the c-Jun/JNK pathway and cyclin-dependent kinases in death of embryonic cortical neurons evoked by DNA damage.

DNA damage, an important initiator of neuronal death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal death evoked by the DNA-damaging agent, camptothecin. In this model, the tumor suppressor p53 and cyclin-dependent kinases (CDKs) are activated independently and cooperate to mediate the conserved death pathway. To further our understanding, we presently examined whether the c-Jun/JNK pathway modulates death and whether this pathway is regulated by CDKs, p53, and Bax. We show that c-Jun/JNK is activated following DNA damage. Moreover, the c-Jun pathway is one mediator of death, because expression of dominant negative c-Jun and cdc42, and JNK pathway inhibitors are neuroprotective. Although previous evidences indicate that JNK3 is required for neuronal death under certain conditions, we show that JNK3 deficiency only partially mediates c-Jun phosphorylation and its deficiency does not protect neurons from death. Interestingly, we provide evidence that CDK activity regulates c-Jun but does not affect upstream pathways that lead to JNK phosphorylation. Finally, c-Jun activation is independent of p53 and Bax. Accordingly, we propose that c-Jun is regulated by the JNK and CDK pathways and that both must be activated for efficient c-Jun activation to occur.

Animals↗

PubMed2003

Ataxia telangiectasia-mutated protein can regulate p53 and neuronal death independent of Chk2 in response to DNA damage.

DNA damage is a key initiator of neuronal death. We have previously shown that the tumor suppressor p53, in conjunction with cyclin-dependent kinases (CDKs), regulates the mitochondrial pathway of death in neurons exposed to genotoxic agents. However, the mechanisms by which p53 is regulated is unclear. Presently, we show that p53 is phosphorylated on Ser-15 following DNA damage and this occurs independently of the CDK pathway. Instead, we show that p53 phosphorylation, stability, as well as neuronal death is regulated, in part, by the ataxia telangiectasia-mutated (ATM) protein. Previous reports have suggested that ATM regulation of p53 occurs through Chk2. However, in our present paradigms, we show that ATM functions separately from Chk2 to regulate p53 stability and neuronal death. Chk2 deficiency does not affect p53 stability or neuronal death induced by Topoisomerase I or II inhibition. Taken together, our results provide a model by which DNA damage can activate an ATM-dependent, Chk2-independent pathway of p53-mediated neuronal death.

Animals↗

PubMed2006

Protein kinase C delta activates topoisomerase IIalpha to induce apoptotic cell death in response to DNA damage.

DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIalpha is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIalpha is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C delta (PKCdelta) interacts with topoisomerase IIalpha. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIalpha in response to DNA damage. Conversely, inhibition of PKCdelta activity attenuates DNA damage-induced activation of topoisomerase IIalpha. Finally, aberrant activation of topoisomerase IIalpha by PKCdelta is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCdelta regulates topoisomerase IIalpha and thereby cell fate in the genotoxic stress response.

Antigens, Neoplasm↗

PubMed2001

Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage.

DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.

Active Transport, Cell Nucleus↗

PubMed2000

Sensing and responding to DNA damage.

DNA damage or stalled DNA replication can activate specific signal transduction pathways, termed checkpoints. Checkpoint activation can result in increased repair, induction of a transcriptional programme and inhibition of cell-cycle progression. Recent results have suggested possible mechanisms for the detection of specific DNA structures, provided further information on the organisation of the signal transduction cascade and demonstrated involvement of the checkpoint pathway in DNA repair.

Animals↗

PubMed1996

Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light-induced DNA damage.

DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage-religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV-induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV-stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA.

Base Sequence↗

PubMed1990

Radiation-induced cytotoxicity, DNA damage and DNA repair: implications for cell survival theory.

The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q greater than 66 Q greater than or equal to 67 P greater than 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.

Animals↗

PubMed2002

DNA topoisomerases in cancer chemotherapy: using enzymes to generate selective DNA damage.

DNA topoisomerase II targeting agents such as etoposide and doxorubicin are well-established cancer chemotherapeutic agents. Topotecan (Hycamtin) and irinotecan (Camptosar) are launched drugs that target topoisomerase I and have significant activity against many solid malignancies. These agents have important mechanistic similarities, converting their target enzyme(s) to generate DNA damage. Recent structural and biochemical studies on targeting of topoisomerases by antitumor agents are providing a framework for understanding drug action at the enzyme level, and at the level of cellular pathways important for responses to this unique type of DNA damage. These investigations into the mechanisms of action of topoisomerase-targeting agents should aid in the design of dinical protocols that optimize the activity of these agents.

Animals↗

PubMed2000

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation.

PURPOSE: To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS: Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS: The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION: It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.

Animals↗

PubMed1995

Regulation of cell cycle progression following DNA damage.

DNA damage causes an arrest in cell cycle progression. Checkpoints, which monitor the state of the DNA, exist throughout the cycle and negatively regulate cell cycle transitions when damage is detected. The molecular basis of how these checkpoints are activated, and interact with the cell cycle machinery, is just beginning to be understood. Studies in yeast have identified a number of genes involved in a G2 DNA damage checkpoint, while in mammalian cells a G1 checkpoint has been extensively studied.

Cell Cycle↗

PubMed1980

Comparison of three in vitro assays for carcinogen-induced DNA damage.

DNA damage induced by five ultimate carcinogens and five procarcinogens was evaluated by three methods: (1) DNA strand breaks in Crandall feline kidney (CRFK) cells measured by alkaline sucrose gradient centrifugation; (2) unscheduled DNA synthesis (UDS) in CRFK, Syrian hamster embryo (SHE), and rat hepatocyte cultures measured by liquid scintillation counting; and (3) UDS in CRFK, SHE, and rat hepatocyte cultures measured by autoradiography. DNA strand breaks were observed with three procarcinogens and four ultimate carcinogens. Only two procarcinogens and two ultimate carcinogens could be identified by liquid scintillation counting. A positive autoradiographic response for UDS was observed in CRFK cells with four of five ultimate carcinogens and one procarcinogen. SHE cells showed a positive autoradiographic response for UDS with all ultimate carcinogens and three procarcinogens. The autoradiographic system with primary rat hepatocyte cultures was the only one in which a positive response for DNA damage was elicited for all 10 carcinogens.

Animals↗

PubMed2003

Lymphocyte DNA damage precedes DNA repair or cell death after orthopaedic surgery under general anaesthesia.

Anaesthetics have gained a lot of attention for their potential mutagenic/carcinogenic effects. In the present study we have investigated the genotoxicity of the inhalation anaesthetic sevoflurane on DNA of lymphocytes isolated from 20 patients undergoing orthopaedic surgery. The genotoxicity of the anaesthetic was studied by assaying DNA damage, apoptosis, DNA repair enzyme activity and GSH content in peripheral lymphocytes before, 15 min after anaesthesia and 24 h after surgery. Lymphocytes isolated 15 min after anaesthesia showed an increase in oxidized purine and pyrimidine bases without DNA strand break formation. DNA strand breaks occurred on the first post-operative day, associated with an enhancement of DNA repair activity and a decrease in GSH. Formation of strand breaks could be the consequence of DNA repair activity. In fact, at 24 h after surgery most of the oxidized DNA bases were repaired. When DNA damage was not repaired, activation of the cell cycle checkpoint protein p53 could lead to apoptosis. An altered redox status may contribute to lymphocytopenia due to an apoptotic event as a consequence of surgical trauma. The presence of apoptotic cells at 1 day after surgery could support the hypothesis that highly damaged peripheral lymphocytes are committed to undergo programmed cell death if the damage is not repaired. In conclusion, the actual risk from anaesthesia is presumably extremely small. However, these findings contribute to our understanding of the regulation of DNA damage/repair and cell death.

Adult↗

PubMed1990

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage.

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.

Alkylating Agents↗

PubMed2005

Evidence for a role of vertebrate Rad52 in the repair of topoisomerase II-mediated DNA damage.

DNA topoisomerase II (Top2) inhibitors are useful as anticancer agents, mostly by virtue of their ability to induce DNA double-strand breaks (DSBs). These DSBs are repaired almost exclusively by Rad52-dependent homologous recombination (HR) in yeast. However, we have recently shown that in vertebrate cells such lesions are primarily repaired by nonhomologous end-joining, but not HR. This finding, taken together with previous observations that disruption of RAD52 does not severely affect HR in vertebrate cells, makes it highly unlikely that Rad52 contributes to the repair of Top2-mediated DNA damage. However, in this paper we show that chicken cells lacking Rad52 do exhibit increased sensitivity to the Top2 inhibitor VP-16. Remarkably, the level of hypersensitivity of RAD52-null cells was comparable to that of RAD54-null cells, albeit only at high doses. Our data thus provide the first demonstration of a major repair defect associated with loss of Rad52 in vertebrate cells.

Animals↗

PubMed2005

Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage.

DNA damage surveillance networks in human cells can activate DNA repair, cell cycle checkpoints and apoptosis in response to fewer than four double-strand breaks (DSBs) per genome. These same networks tolerate telomeres, in part because the protein TRF2 prevents recognition of telomeric ends as DSBs by facilitating their organization into T loops. We now show that TRF2 associates with photo-induced DSBs in nontelomeric DNA in human fibroblasts within 2 s of irradiation. Unlike gammaH2AX, a common marker for DSB damage, TRF2 forms transient foci that colocalize closely with DSBs. The TRF2 DSB response requires the TRF2 basic domain but not its Myb domain and occurs in the absence of functional ATM and DNA-PK protein kinases, MRE11/Rad50/NBS1 complex and Ku70, WRN and BLM repair proteins. Furthermore, overexpression of TRF2 inhibits DSB-induced phosphorylation of ATM signaling targets. Our results implicate TRF2 in an initial stage of DSB recognition and processing that occurs before association of ATM with DSBs and activation of the ATM-dependent DSB response network.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage.

DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.

Animals↗

PubMed2003

Activity of megazol, a trypanocidal nitroimidazole, is associated with DNA damage.

DNA damage associated with the trypanocidal activity of megazol [2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole] was shown in experiments in which DNA repair-deficient RAD51(-/-) Trypanosoma brucei mutants were found to be hypersensitive to the drug. Parasites resistant to megazol were selected and showed modest cross-resistance to other trypanocides, although neither drug efflux nor changes to intracellular thiols correlated with resistance.

Animals↗