2-Naphthol Moiety of Neocarzinostatin Chromophore as a Novel Protein-Photodegrading Agent and Its Application as a H2 O2 -Activatable Photosensitizer.

A 2-naphthol derivative 2 corresponding to the aromatic ring moiety of neocarzinostatin chromophore was found to degrade proteins under photo-irradiation with long-wavelength UV light without any additives under neutral conditions. Structure-activity relationship studies of the derivative revealed that methylation of the hydroxyl group at the C2 position of 2 significantly suppressed its photodegradation ability. Furthermore, a purpose-designed synthetic tumor-related biomarker, a H2 O2 -activatable photosensitizer 8 possessing a H2 O2 -responsive arylboronic ester moiety conjugated to the hydroxyl group at the C2 position of 2, showed significantly lower photodegradation ability compared to 2. However, release of the 2 from 8 by reaction with H2 O2 regenerated the photodegradation ability. Compound 8 exhibited selective photo-cytotoxicity against high H2 O2 -expressing cancer cells upon irradiation with long-wavelength UV light.

Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase.

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3'-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3'-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP -/- HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3'-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3'-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3'-phosphate-terminated and fewer 3'-hydroxyl-terminated DSBs than parental cells 5-15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3'-phosphatase, loss of PNKP significantly sensitizes cells to 3'-phosphate-terminated DSBs, due to a 3'-dephosphorylation defect.

DNA Damage Detection by 53BP1: Relationship to Species Longevity.

In order to examine potential differences in genomic stability, we have challenged fibroblasts derived from five different mammalian species of variable longevity with the genotoxic agents, etoposide and neocarzinostatin. We report that cells from longer-lived species exhibit more tumor protein p53 binding protein 1 (53BP1) foci for a given degree of DNA damage relative to shorter-lived species. The presence of a greater number of 53BP1 foci was associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. These data suggest that cells from longer-lived species have an enhanced DNA damage response. We propose that the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms.

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3'-phosphoglycolate termini in SCAN1 cells.

To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3'-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3'-phosphate and 3'-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3'-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3'-PG and 3'-phosphate termini on 1-base 3' overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3'-PG termini were processed more slowly than 3'-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3'-PG blocking groups for DSB repair. DSBs with 3'-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3'-PG ends.

DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway.

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.

Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis.

Deficiency in Artemis is associated with lack of V(D)J recombination, sensitivity to radiation and radiomimetic drugs, and failure to repair a subset of DNA double-strand breaks (DSBs). Artemis harbors an endonuclease activity that trims both 5'- and 3'-ends of DSBs. To examine whether endonucleolytic trimming of terminally blocked DSBs by Artemis is a biologically relevant function, Artemis-deficient fibroblasts were stably complemented with either wild-type Artemis or an endonuclease-deficient D165N mutant. Wild-type Artemis completely restored resistance to γ-rays, bleomycin and neocarzinostatin, and also restored DSB-repair proficiency in G0/G1 phase as measured by pulsed-field gel electrophoresis and repair focus resolution. In contrast, cells expressing the D165N mutant, even at very high levels, remained as chemo/radiosensitive and repair deficient as the parental cells, as evidenced by persistent γ-H2AX, 53BP1 and Mre11 foci that slowly increased in size and ultimately became juxtaposed with promyelocytic leukemia protein nuclear bodies. In normal fibroblasts, overexpression of wild-type Artemis increased radioresistance, while D165N overexpression conferred partial repair deficiency following high-dose radiation. Restoration of chemo/radioresistance by wild-type, but not D165N Artemis suggests that the lack of endonucleolytic trimming of DNA ends is the principal cause of sensitivity to double-strand cleaving agents in Artemis-deficient cells.

XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.

Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery.

BACKGROUND: The cytotoxicity and the rejoining of DNA double-strand breaks induced by gamma-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair. RESULTS: PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by gamma-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following gamma-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as gamma-rays and H2O2. CONCLUSIONS: The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to gamma-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

Gene rearrangements induced by the DNA double-strand cleaving agent neocarzinostatin: conservative non-homologous reciprocal exchanges in an otherwise stable genome.

Among a collection of 74 aprt mutations induced by treatment of plateau phase Chinese hamster ovary CHO cells with the radiomimetic DNA double-strand cleaving agent neocarzinostatin, nine were large-scale rearrangements. Molecular analysis indicated that all nine were highly conservative, non-homologous reciprocal exchanges, most of which were intrachromosomal as determined by fluorescence in situ hybridization. All but one of the parental sequences contained potential double-strand cleavage sites positioned such that the observed rearrangements could be explained by drug-induced double-strand breakage followed by trimming, templated patching and ligation of the exchanged ends. Predicted non-complementary 3' overhangs were often preserved in the newly formed junctions, suggesting alignment-based fill-in of the overhangs. Banding of metaphase spreads of these mutants, and of a number of mutants induced by the functionally similar compound bleomycin, revealed that bleomycin-induced reciprocal exchange mutants had multiple additional chromosome alterations and considerable chromosomal heterogeneity within each mutant line. In contrast, neocarzinostatin-induced reciprocal exchange mutants, as well as bleomycin-induced base substitution and single base deletion mutants, retained stable pseudodiploid karyotypes similar to that of the parent line. Thus, some reciprocal exchanges arising from misjoining of double-strand breaks were associated with global chromosomal instability, while other ostensibly similar events were not.

Delocalized electronic structure of the thiol sulfur substantially prevents nucleic acid damage induced by neocarzinostatin.

Neocarzinostatin is a potent antitumor antibiotic and is a prodrug, which induces genome damage after activation by a thiol. The prodrug is stored as a protein-bound chromophore that contains an enediyne nucleus. A thiolate attack on the chromophore cyclizes the nucleus and produces radicals that abstract hydrogen from DNA. Because thiol is the only cofactor in the vital activation process, the structure of the thiol plays an important role in the activity of the drug. Here we systematically examine the effect of the electronic structure of some thiols on the efficiency of the drug, and compare particularly aromatic with aliphatic thiols. The values of drug-induced base release from DNA are remarkably different between thiophenol (3.6%) and benzyl mercaptan (12.5%), the activity of which is comparable with those of aliphatic thiols. Cleavage results determined by DNA electrophoresis are consistent with the results of base release; they show that the total number of DNA lesions is more than 3-fold lower for thiophenol than for aliphatic thiols or benzyl mercaptan. We conclude that among aromatic thiols, only those that have delocalized thiol sulfur electrons can substantially reduce the DNA cleavage activity. This result suggests that the effect of an aromatic ring arises from an inductive effect imposed on the thiol sulfur electron through pi-resonance rather than through effects such as aromatic stacking, steric hindrance, or hydrophobic interaction. Replacing thiophenol with substituted derivatives with electron-releasing or -withdrawing groups changes the drug activity and supports the important role of the electronic structure of the thiol sulfur in determining the drug activity.

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication.

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.

Differential responses of Chinese hamster mutagen sensitive cell lines to low and high concentrations of calicheamicin and neocarzinostatin.

To shed light on the mechanism underlying the cellular response to the radiomimetic agents calicheamicin Y(1)(1) (CAL) and neocarzinostatin (NCS), several hamster cell mutants defective in different DNA repair pathways were used. Two X-ray sensitive Chinese hamster V79 mutant cell lines, XR-V9B and V-E5 were studied for their response to the induction of cell killing, micronuclei, and G2-chromosomal aberrations relative to that of parental wild-type cells. In addition, effects of CAL and NCS on bleomycin sensitive BL-V40 cells and on UV sensitive V-H1 cells were analyzed. In general, the radiosensitive cell lines showed the highest sensitivities to CAL and NCS, but also the other mutants demonstrated differences in their responses compared to wild-type cells. With respect to cell killing, expressed as D(10)-value, enhanced sensitivities of mutants with factors up to 4.4 were recorded. For the induction of micronuclei (MN) and chromosomal aberrations (CA) all cell lines, including the parental cells, show a steep increase in the frequencies at the lowest tested doses and a leveling off at higher concentrations. Probably toxic effects at the higher exposure levels are responsible for these biphasic dose effect curves. Enhanced sensitivities of the various mutants were primarily observed at the higher exposure levels. With respect to the induction of MN increased sensitivities up to a factor of 18.1 were observed for the radiosensitive mutants, whereas for CA the mutant cell lines showed a variation from resistance (0.3) of VH-1 cells up to a 3.8-fold higher sensitivity to the radiomimetic agents. However, at the lowest tested concentrations for both MN and CA, the differences between the sensitive mutants and wild-type clearly diminished, suggesting the existence of residual and/or alternative DNA repair pathways in these mutants.

Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation.

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.

Thiopronin reduces the frequencies of neocarzinostatin-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were treated in the G1 phase of the cell cycle with different concentrations of neocarzinostatin (NCS) alone or in combination with N-(2-mercaptopropionyl)-glycine (thiopronin; TP). TP reduces the frequencies of NCS-induced chromosomal aberrations (CA) and of sister chromatid exchanges (SCE) significantly when added to the cultures simultaneously (TPsim), 1 min (TP1) or 10 min (TP10) after the addition of NCS. The addition of TP 30 min (TP30) or 60 min (TP60) after NCS reduces the frequencies of SCE, but not of CA. Our results indicate that the induction of CA and SCE by NCS is partially based on different mechanisms.

DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay.

Using the alkaline comet assay (single cell gel electrophoresis), we studied the induction and persistence of DNA damage induced by methyl methanesulfonate (MMS) and neocarzinostatin (NCS) in the repair-deficient Chinese hamster cell lines V-E5 and XR-V15B. Effects in the comet assay were analyzed directly after treatment as well as after a postincubation period in mutagen-free medium to gain insight into the DNA repair capacities of the mutant cell lines in relation to different primary DNA lesions. Both mutagens caused a concentration-related increase in DNA strand breakage in both mutant cell lines and in the normal parental cell lines. Repair of MMS-induced DNA damage during postincubation was similar in normal and mutant cell lines, while diminished repair was seen after NCS treatment in XR-V15B cells. Our data show that XR-V15B cells only repaired about 30% of NCS-induced DNA damage within 1 h, while the parental V79 cell line repaired about 70%. Since this cell line is defective in the repair of DNA double-strand breaks (DSB), the results indicate that NCS-induced DSB significantly contribute to the genotoxic effects seen in the comet assay. However, compared to previously studied induction of gene mutations and chromosome aberrations, detection of NCS-induced DNA effects with the comet assay was less sensitive and increased DNA migration only occurred under strong cytotoxic conditions.

Micronucleus induction by neocarzinostatin and methyl methanesulfonate in ionizing radiation--sensitive Chinese hamster V79 cell mutants.

Two mutant V79 Chinese hamster cell lines (V-E5, XR-V15B) which show hypersensitivities to DNA damage and their two parental cell lines (V79-LE, V79-B) were used for micronucleus studies. The characteristics of V-E5 strongly resemble those of cells derived from patients suffering from the genomic instability syndrome ataxia telangiectasia, whereas XR-V15B has a decreased ability to rejoin double-strand breaks. The two cell lines V-E5 and XR-V15B showed increased spontaneous micronucleus frequencies and higher sensitivity for micronucleus induction by methyl methanesulfonate (MMS) and neocarzinostatin (NCS) both with and without the use of cytochalasin B in the micronucleus assay. Thus, defects in cellular responses to DNA damage are modulating factors in micronucleus formation.

Mechanism of free and conjugated neocarzinostatin activity: studies on chromophore and protein uptake using a transferrin-neocarzinostatin conjugate.

Targeting studies using the anti-cancer agent neocarzinostatin (NCS), conjugated to anti-bodies have shown relatively poor specificity. From the literature, it is unclear whether NCS mediates its effects either in conjugated or unconjugated form. In the present work we have used a conjugate of NCS with transferrin, a biological ligand with a well defined endocytic route, to probe these mechanisms. NCS was covalently coupled to transferrin using the heterobifunctional reagent sulfo-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 2-iminothiolane to give a stable thioether-linked conjugate with a ratio of 1.6 mol of NCS per mole of transferrin. The binding activity of transferrin was completely retained. Conjugation of NCS to transferrin resulted in an apparent enhancement of cytotoxicity. However, incubation with excess transferrin had no influence on the observed enhanced toxicity, indicating that endocytosis is not responsible. Further experiments demonstrated that the apparent enhancement was dependent on incubation conditions and not an effect due to endocytosis of ligand. Studies where apo-NCS competed with holo-NCS and transferrin strongly indicated that the cytotoxicity of both NCS and conjugate is mediated by direct entry of the dissociated chromophore into the cell.

DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes.

Bleomycin and the enediyne antibiotics effect concerted, simultaneous site-specific free radical attack on sugar moieties in both strands of DNA, resulting in double-strand breaks of defined geometry and chemical structure, as well as abasic sites with closely opposed strand breaks. The hypersensitivity of several mammalian double-strand break repair-deficient mutants to these agents confirms the role of these double-strand breaks in mediating cytotoxicity. In bacteria, mutagenesis by both bleomycin and neocarzinostatin appears to result from replicative bypass of abasic sites, the repair of which is blocked by the presence of closely opposed strand breaks. However, in mammalian cells, such abasic sites decompose to form double-strand breaks, and mutagenesis consists primarily of small deletions, large deletions, and gene rearrangements, all of which probably result from errors in double-strand break repair by a nonhomologous end-joining mechanism. Studies with the radiomimetic antibiotics emphasize the importance of this end-joining repair pathway, and these agents provide useful probes of its mechanistic details, particularly the effects of chemically modified DNA termini on repair.

Detection of neocarzinostatin-induced translocations in human sperm chromosomes using fluorescence in situ hybridization of chromosome 2.

Mature sperm and late spermatid are known to be sensitive stages to clastogens in mammalian spermatogenesis. Certain types of chromosomal damage induced in these stages will pass to successive generations as heritable translocations. In the present study, we employed whole chromosome 2 painting with the fluorescence in situ hybridization (FISH) technique to detect the chemically induced translocations in human sperm. Mature human sperm were treated in vitro with an antitumor drug, neocarzinostatin (NCS), and fertilized in vitro with golden hamster oocytes. Sperm pronuclear chromosome slides were prepared at the first cleavage metaphase. To compare the characteristics of translocations between somatic and germ cells, human lymphocytes in peripheral blood treated with NCS in vitro were analyzed at first round metaphase after PHA-stimulation. From the analysis of translocations by whole chromosome 2 painting, frequencies of the haploid genomic translocations (FhG) were predicted for both sperm and lymphocytes. At 1.0 micrograms/ml, the actual percentages of sperm and lymphocytes with chromosome 2 translocations were almost identical (11.9% and 12.0%). At the same dose, however, the FhG of the sperm (1.15) was considerably higher than that of the lymphocytes (0.58), indicating that complex translocations having two or more rearranged sites were induced by NCS more frequently in sperm than in lymphocytes.