RESUMEN
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.
Asunto(s)
Naftoles , Proteínas , Cinostatina/análogos & derivados , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/química , Ratones , Naftoles/química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/toxicidad , Proteínas/efectos de los fármacos , Proteínas/efectos de la radiación , Cinostatina/química , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Glicolatos/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Línea Celular Transformada , ADN/química , Proteína Quinasa Activada por ADN/deficiencia , Humanos , Hidrolasas Diéster Fosfóricas/deficiencia , Ataxias Espinocerebelosas/genética , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Endodesoxirribonucleasas/genética , Proteínas Nucleares/genética , Tolerancia a Radiación , Bleomicina/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Proteínas de Unión al ADN , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/metabolismo , Endonucleasas , Fase G1 , Humanos , Mutación , Proteínas Nucleares/análisis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Ligasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Células Cultivadas , Cricetinae , ADN Ligasa (ATP) , Etopósido/toxicidad , Humanos , Ratones , Cinostatina/toxicidadRESUMEN
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 5-15â¯min after NCS treatment, but this difference disappeared by 1â¯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.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ADN/efectos de los fármacos , ADN/metabolismo , ADN/efectos de la radiación , Enzimas Reparadoras del ADN/metabolismo , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HeLa , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Radiación Ionizante , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Daño del ADN , Fibroblastos/metabolismo , Longevidad , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Bovinos , Puntos de Control del Ciclo Celular , Línea Celular , Quirópteros , Ciclina A/metabolismo , Citotoxinas/toxicidad , Fragmentación del ADN , Perros , Etopósido/toxicidad , Fibroblastos/efectos de los fármacos , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Esperanza de Vida , Ratones , Micronúcleos con Defecto Cromosómico , Pruebas de Micronúcleos , Quinasas Relacionadas con NIMA/metabolismo , Inhibidores de Topoisomerasa II/toxicidad , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Daño del ADN , Recombinación Genética , Cinostatina/toxicidad , Adenina Fosforribosiltransferasa/genética , Animales , Secuencia de Bases , Células CHO , Aberraciones Cromosómicas , Cricetinae , Genoma , Datos de Secuencia Molecular , Mutágenos/toxicidad , MutaciónRESUMEN
The effects of environmental pH on the binding and cytotoxicity of the antitumor proteins neocarzinostatin (NCS) and SMANCS [copoly(styrene-maleic acid)-conjugated NCS] to cultured cells were studied by using their fluorescent-labeled derivatives (F-drugs). At 37 degrees C the binding of these drugs to HeLa cells was pH dependent: The amount of cell-bound drugs increased with an increase in the acidity of the medium. The pH-dependent change in the binding of the drugs was not as evident at 0 degree C. The cytotoxic action of these drugs was much more rapid at acidic pH compared with that at neutral or slightly alkaline pH. Furthermore, F-drugs could be utilized to probe the microenvironmental pH in Meth-A cells, in which the drug was located by the ratio of fluorescent intensities at 450 and 490 nm. The environment of the cell-bound F-drugs became acidic with incubation time at 37 degrees C but not at 0 degree C. Inasmuch as these drugs directly attack DNA, these results suggest that NCS and SMANCS are translocated across the membrane of acidic vesicles into the cytosol after endocytotic uptake. This hypothesis is also supported by the finding that NH4Cl and chloroquine protected HeLa cells against the cytotoxicity of the drugs. Data also showed that the hydrophobic polyanion conjugate SMANCS had a much greater cell binding (10 times) and more rapid internalization compared with NCS. Taken together, our results show that acidic pH of tumor tissue is preferable for effective binding and internalization into cytosol for NCS and SMANCS.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Furanos/metabolismo , Anhídridos Maleicos/metabolismo , Poliestirenos/metabolismo , Cinostatina/metabolismo , Cloruro de Amonio/farmacología , Animales , Células Cultivadas , Cloroquina/farmacología , Citosol/metabolismo , Fluoresceína-5-Isotiocianato , Fluoresceínas , Colorantes Fluorescentes , Células HeLa/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Anhídridos Maleicos/toxicidad , Ratones , Ratones Endogámicos BALB C , Poliestirenos/toxicidad , Unión Proteica , Tiocianatos , Cinostatina/análogos & derivados , Cinostatina/toxicidadRESUMEN
Neocarzinostatin (NCS) is mutagenic in bacteria, yeast, fungi, and mammalian cells. In cell-free systems, DNA strand breakage induced by NCS requires a reducing agent like 2-mercaptoethanol, unless very high (greater than 100 micrograms/ml) concentrations of NCS are used. In this study, we have investigated the role of the sulfhydryl compound glutathione (GSH), which is usually the most common intracellular thiol, in the bioactivation of NCS to a toxic and mutagenic species. Chinese hamster V79 cells were pretreated with one of two GSH depleting agents, buthionine sulfoximine or diethyl maleate. These agents deplete GSH via different mechanisms, but both will lower GSH levels within the cell to less than 5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5-2.5 micrograms/ml for 1 h, assayed for survival, and plated for expression of hypoxanthine-guanine phosphoribosyltransferase-negative (HGPRT-) mutants. After an expression period of 7 days, during which the cultures were subcultured twice, HGPRT- mutants were selected by plating in hypoxanthine-free medium containing 5 micrograms of 6-thioguanine per ml, at a density of 2 X 10(5) cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 micrograms/ml and produced dose-related increases in HGPRT-mutants that reached greater than 10 times the spontaneous mutation frequency at 2.5 micrograms NCS per ml. In GSH-depleted cells, however, NCS was only mildly cytotoxic (60-80% surviving fraction) and did not produce dose-related increases in HGPRT- mutants over cells treated only with diethyl maleate or buthionine sulfoximine. Thus, GSH appears to be the main reducing agent for the bioactivation of NCS to a toxic and mutagenic species in Chinese hamster V79 cells.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Glutatión/fisiología , Mutágenos , Cinostatina/toxicidad , Animales , Biotransformación , Butionina Sulfoximina , Cricetinae , Cricetulus , Maleatos/farmacología , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Cinostatina/metabolismoRESUMEN
An alkaline elution procedure was used to study the nature of DNA damage induced by auromomycin, an antitumor protein, in human leukemic lymphoblasts (CCRF-CEM cells). The filter elution of drug-treated cells at pH 12.2 and 9.6 showed induction of both single and double strand DNA breaks. The DNA strand scission activities were linear in relation to drug concentration. The frequency of single strand breaks was higher than that of the double strand breaks. Protein-associated DNA single strand breaks were also detected in alkaline elution of drug-treated cells when a proteinase K digestion step was included in the assay protocol. The auromomycin-induced single strand breaks were repaired to almost completion within 8 h of postincubation of DNA-damaged cells whereas the repair of double strand breaks was not detected.
Asunto(s)
Antibacterianos , Antibióticos Antineoplásicos/toxicidad , Reparación del ADN/efectos de los fármacos , Apoproteínas/toxicidad , Línea Celular , ADN de Neoplasias/genética , ADN de Neoplasias/efectos de la radiación , Endopeptidasa K , Endopeptidasas/metabolismo , Rayos gamma , Humanos , Leucemia Linfoide , Peso Molecular , Péptidos/toxicidad , Relación Estructura-Actividad , Cinostatina/toxicidadRESUMEN
The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Cinostatina/toxicidad , Animales , Radioisótopos de Carbono , Línea Celular , Cricetinae , Cricetulus , ADN/genética , ADN/efectos de la radiación , Femenino , Cinética , Mutación , OvarioRESUMEN
We previously found that a high-molecular-weight anticancer agent, polystyrene-co-maleic acid conjugated neocarzinostatin (SMANCS), in which two chains of styrene/maleic acid copolymer are conjugated to the anticancer protein neocarzinostatin (NCS), accumulated more selectively in tumor tissue than in normal tissue and was more stable than NCS in blood. These results indicate that SMANCS should have less systemic toxicity and a better therapeutic effect than NCS. In this study, the antitumor activity and adverse effects of SMANCS were compared with those of NCS by using rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene. When tumors of rats, that had received 7,12-dimethylbenz[a]anthracene (20 mg/kg, one dose, p.o. in oily formulation), became palpable usually after 4-20 weeks, SMANCS treatment was initiated. Thirty days after i.v. administration of SMANCS (0.1 mg/kg 3 times and 0.3 mg/kg 3 times), tumors had shrunk in 35 of 37 rats (a mean weight was about 10% of control value; or decreased to about 30% of the value of before treatment in tumor weight); tumor size had not changed in 1 rat, and in the remaining 1 rat the tumor had enlarged. Thirty days after i.v. administration of NCS, tumors had shrunk in 8 of 14 rats, but the tumor size was unchanged in 1 rat and was enlarged in 5. In the control group, all tumors had enlarged. Development of new tumors was completely prevented by the administration of SMANCS. Histological examination of sequential slices of tumor revealed clear finding of degeneration and tumor encapsulation at 30 days after initial administration of SMANCS, with an accompanying fatty degeneration, but these effects were not observed for tumors treated with NCS. Although red blood cell counts and hemoglobin amounts decreased significantly in rats receiving NCS, no such effects were apparent in the SMANCS group.
Asunto(s)
Anhídridos Maleicos/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Poliestirenos/uso terapéutico , Cinostatina/análogos & derivados , 9,10-Dimetil-1,2-benzantraceno , Animales , División Celular , Recuento de Eritrocitos/efectos de los fármacos , Femenino , Hemoglobinas/metabolismo , Anhídridos Maleicos/toxicidad , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/patología , Recuento de Plaquetas/efectos de los fármacos , Poliestirenos/toxicidad , Ratas , Ratas Endogámicas , Cinostatina/uso terapéutico , Cinostatina/toxicidadRESUMEN
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.
Asunto(s)
Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Daño del ADN , Técnica del Anticuerpo Fluorescente , Rayos gamma , Peróxido de Hidrógeno/toxicidad , Ratones , Ratones Noqueados , Células 3T3 NIH , Poli(ADP-Ribosa) Polimerasas/genética , Cinostatina/toxicidadRESUMEN
The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , ADN/metabolismo , Mutágenos , Cinostatina/toxicidad , Animales , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Antibacterianos/toxicidad , Antibióticos Antineoplásicos/toxicidad , Liasas de Carbono-Oxígeno/metabolismo , Daño del ADN , ADN/metabolismo , Proteínas de Escherichia coli , Exodesoxirribonucleasas/metabolismo , Glicolatos , Cinostatina/toxicidad , Aminoglicósidos , Animales , Bovinos , ADN/efectos de los fármacos , ADN/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/enzimología , Rayos gamma , Humanos , CinéticaRESUMEN
The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible changes in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS.
Asunto(s)
Aberraciones Cromosómicas , Reparación del ADN , ADN/genética , Mutación , Animales , Línea Celular , Cricetinae , Cricetulus , ADN/efectos de los fármacos , Reparación del ADN/genética , Hipoxantina Fosforribosiltransferasa/genética , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , Reacción en Cadena de la Polimerasa , Cinostatina/toxicidadRESUMEN
Chinese hamster ovary cells were treated with hypertonic 0.5 M NaCl solution after exposure to X rays or the radiomimetic drugs bleomycin or neocarzinostatin. The cytotoxicity of these agents was greatly enhanced by the hypertonic treatment. On the other hand, no effect was observed after exposure to ultraviolet light, and a significant effect was obtained with mitomycin C (MMC), adriamycin (ADR), and ethyl methanesulfonate (EMS). Assays by filter elution revealed that hypertonicity had various effects on the damage produced by the different agents. Strand breaks resulting from exposure to X rays and radiomimetic agents were sensitive to hypertonic treatment. Hypertonicity caused the production of new lesions and inhibited the rejoining of DNA strand breaks, both of which may be responsible for the enhanced cytotoxicity. On the other hand, the formation of crosslinks by MMC or protein-associated double-strand breaks by ADR, the major forms of damage by which these agents cause cytotoxicity, was not affected by hypertonic treatment. As strand breaks are known to be produced by MMC or ADR, they could account at least partly for the sensitization. However, various kinds of damage are produced by MMC, and any of these could be involved in the sensitization. To our knowledge EMS produces only base damage. Thus hypertonic treatment may have an effect on various types of damage.
Asunto(s)
Bleomicina/toxicidad , Daño del ADN , Reparación del ADN/efectos de los fármacos , Soluciones Hipertónicas/farmacología , Cinostatina/toxicidad , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cricetinae , Daño del ADN/efectos de los fármacos , Concentración Osmolar , Rayos XRESUMEN
We studied the effect of "two-route chemotherapy" (TRC) with intra-arterial (IA) neocarzinostatin (NCS) and IV N-(2-mercaptopropionyl)-glycine (tiopronin), its antidote, on rat limb tumors. Chemotherapy experiments were carried out on day 9 after the inoculation of 10(6) syngeneic transitional carcinoma cells into the hind limb in female Wistar King A rats. In the group given TRC, 3500 units/kg NCS and 800 mg/kg tiopronin were given via the femoral artery and the femoral vein, respectively. The antitumor effect was evaluated by the tumor weight on day 12 after the treatment. Compared with the weight of tumors in untreated controls, TRC reduced tumor weight to one-tenth, while 700 units/kg IA NCS alone reduced tumor weight to one-third and 700 units/kg systemic NCS alone reduced tumor weight to three-fourths of the control weight. In the group given TRC, WBC and nucleated bone marrow cells were completely protected and loss of body weight was slight.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma de Células Transicionales/tratamiento farmacológico , Animales , Peso Corporal/efectos de los fármacos , Médula Ósea/metabolismo , Femenino , Infusiones Intraarteriales , Infusiones Parenterales , Leucocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Ratas , Ratas Endogámicas , Tiopronina/administración & dosificación , Cinostatina/administración & dosificación , Cinostatina/toxicidadRESUMEN
The cytoxicity of neocarzinostatin (NCS) and smancs [copoly(styrene maleic acid)-conjugated NCS] to various cultured cells was compared with that of several other antitumor agens in clinical use on various malignant and non-malignant cells as regards to their effect on colony formation of cells. Both NCS and smancs showed the most potent cytotoxicity against all tumor cell lines tested; the IC50s (colony inhibitory concentration 50%) of these drugs were 3.2-20 nM, 10-1000 times lower than those of other drugs. In contrast, NCS and smancs exhibited relatively lower toxicity to normal cells such as human skin fibroblasts and chick embryonic fibroblasts (IC50, about 50 and 100 nM, respectively). Normal rat hepatocytes were found to be very resistant to NCS and smancs (both IC50s were about 500 nM). Moreover, the minimum exposure time of smancs to cultured tumor cells required to achieve effective cytotoxic activity was much shorter than that of NCS and other drugs. Namely, at 30 nM more than 80% cells were killed by exposure to smancs for only a few minutes, whereas with NCS more than 80 min of exposure time was required. It was also found that smancs inhibited the uptake of 3H-thymidine into DNA as expected. These results clearly indicate that smancs is an unique antitumor agent with a broad antitumor spectrum which exhibits some characteristics similar to, but also some very different from NCS.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Furanos/toxicidad , Anhídridos Maleicos/toxicidad , Poliestirenos/toxicidad , Cinostatina/toxicidad , Animales , Antineoplásicos/toxicidad , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Ratones , Cinostatina/análogos & derivadosRESUMEN
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.