[Show abstract][Hide abstract] ABSTRACT: Loss of function of DNA repair (DNAR) genes is associated with genomic instability and cancer predisposition; it also makes cancer cells reliant on a reduced set of DNAR pathways to resist DNA-targeted therapy, which remains the core of the anticancer armamentarium. Because the landscape of DNAR defects across numerous types of cancers and its relation with drug activity have not been systematically examined, we took advantage of the unique drug and genomic databases of the US National Cancer Institute cancer cell lines (the NCI-60) to characterize 260 DNAR genes with respect to deleterious mutations and expression down-regulation; 169 genes exhibited a total of 549 function-affecting alterations, with 39 of them scoring as putative knockouts across 31 cell lines. Those mutations were compared to tumor samples from 12 studies of The Cancer Genome Atlas (TCGA) and The Cancer Cell Line Encyclopedia (CCLE). Based on this compendium of alterations, we determined which DNAR genomic alterations predicted drug response for 20,195 compounds present in the NCI-60 drug database. Among 242 DNA damaging agents, 202 showed associations with at least one DNAR genomic signature. In addition to SLFN11, the Fanconi anemia-scaffolding gene SLX4 (FANCP/BTBD12) stood out among the genes most significantly related with DNA synthesis and topoisomerase inhibitors. Depletion and complementation experiments validated the causal relationship between SLX4 defects and sensitivity to raltitrexed and cytarabine in addition to camptothecin. Therefore, we propose new rational uses for existing anticancer drugs based on a comprehensive analysis of DNAR genomic parameters.
Published by Elsevier B.V.
[Show abstract][Hide abstract] ABSTRACT: The use of mesenchymal stem cells (MSCs) in experimental, clinical, and therapeutic trials has grown in recent years. However, the issue remains of whether these procedures are completely safe for transplant patients. Therefore, this study was designed and carried out with the aim of evaluating two different comet assay protocols for genomic damage pattern analysis in MSCs derived from adipose tissue. The analyzed and interpreted results suggest that genetic testing is needed to support clonal expansion safety in cell therapy procedures with MSCs. Furthermore, they also suggest that if the comet assay technique would be used as a genomic integrity screening assay, the protocol performed at pH = 12 (that yielded a frequency of damaged cells: tail intensity = 9.50 ± 0.60, tail moment = 0.0122 ± 0.0007; results are reported as means ± standard deviation) would be indicated as genomic damage, and that subsequent single-strand breaks occur at pH > 13 (frequency of damaged cells: tail intensity = 30.71 ± 4.23, tail moment = 0.0447 ± 0.0073). Our study demonstrates that, in the era of regenerative medicine, it is necessary to standardize and establish a battery of tests in order to identify genomic damage prior to MSC transplantation.
Full-text · Article · Jan 2015 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: Rupture of the Achilles tendon diminishes quality of life. The gold-standard therapy is a surgical suture, but this presents complications, including wound formation and inflammation. These complications spurred evaluation of the therapeutic potential of mesenchymal stem cells (MSCs) from adipose tissue. New Zealand rabbits were divided into 6 groups (three treatments with two time points each) evaluated at either 14 or 28 days after surgery: cross section of the Achilles tendon (CSAT); CSAT + Suture; and CSAT + MSC. A comparison between all groups at both time points showed a statistically significant increase in capillaries and in the structural organization of collagen in the healed tendon in the CSAT + Suture and CSAT + MSC groups at the 14-day assessment. Comparison between the two time points within the same group showed a statistically significant decrease in the inflammatory process and an increase in the structural organization of collagen in the CSAT and CSAT + MSC groups. A study of the genomic integrity of the cells suggested a linear correlation between an increase of injuries and culture time. Thus, MSC transplantation is a good alternative for treatment of Achilles tendon ruptures because it may be conducted without surgery and tendon suture and, therefore, has no risk of adverse effects resulting from the surgical wound or inflammation caused by nonabsorbable sutures. Furthermore, this alternative treatment exhibits a better capacity for wound healing and maintaining the original tendon architecture, depending on the arrangement of the collagen fibers, and has important therapeutic potential.
Full-text · Article · Dec 2014 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: We evaluated the effects of glutamine on clastogenic and genotoxic damage prevention caused by the administration of cisplatin. Forty Swiss mice were divided into 8 experimental groups: G1 and G2, which were control groups; G3, G4, and G5, which were administered [2 doses of glutamine (orally)] separated by a 24-h period (150, 300, and 600 mg/kg, respectively), and a dose of phosphate-buffered saline by intraperitoneal injection; G6, G7, and G8, which were treated in the same manner as the previous groups, but received cisplatin rather than phosphate-buffered saline. The antimutagenicity groups showed damage reduction percentages of 79.05, 80.00, and 94.27% at the time point T1, 53.18, 67.05, and 64.74 at time point T2 for the 150, 300, and 600 mg/kg doses of glutamine, respectively. Antigenotoxic activity was evident for all 3 doses with damage reduction percentages of 115.05, 119.06, and 114.38 for the doses of glutamine of 150, 300, and 600 mg/ kg, respectively. These results suggest that further studies are needed to confirm the clastogenic activity of glutamine. However, our results may lead to rational strategies for supplementation of this antioxidant as an adjuvant in cancer treatment or for preventing genomic lesions.
[Show abstract][Hide abstract] ABSTRACT: Tyrosyl-DNA-phosphodiesterase 1 (TDP1) repairs 3'-blocking DNA lesions by catalytically hydrolyzing the tyrosyl-DNA-phosphodiester bond of trapped topoisomerase I (Top1) cleavage complexes (Top1cc). It also removes 3'-blocking residues derived from oxidative damage or incorporation of chain terminating anticancer and antiviral nucleosides. Thus, TDP1 is regarded as a determinant of resistance to Top1 inhibitors and chain terminating nucleosides, and possibly of genomic stability. In the 60 cell lines of the NCI Developmental Therapeutic Anticancer Screen (the NCI-60), whose whole genome transcriptome and mutations have recently been characterized, we discovered two human lung cancer cell lines deficient for TDP1 (NCI_H522 and HOP_62). HOP_62 shows undetectable TDP1 mRNA and NCI_H522 bears a homozygous deleterious mutation of TDP1 at a highly conserved amino acid residue (K292E). Absence of TDP1 protein and lack of TDP1 catalytic activity were demonstrated in cell lysates from both cell lines. Lack of TDP1 expression in HOP_62 was shown to be due to TDP1 promoter hypermethylation. Our study provides insights into the possible inactivation of TDP1 in cancers and its relationship to cellular response to Top1-targeted drugs. It also reveals two TDP1 knockout lung cancer cell lines for further TDP1 functional analyses.
[Show abstract][Hide abstract] ABSTRACT: Cisplatin is an effective antineoplastic drug. However, it provokes considerable collateral effects, including genotoxic and clastogenic activity. It has been reported that a diet rich in glutamine can help inhibit such collateral effects. We evaluated this activity in 40 Swiss mice, distributed into eight experimental groups: G1 - Control group (PBS 0.1 mL/10g body weight); G2 - cisplatin group (cisplatin 6 mg/kg intraperitoneally); G3, G4, G5 - glutamine groups (glutamine at 150, 300, and 600 mg/kg, respectively; orally); G6, G7, G8 - Pre-treatment groups (glutamine at 150, 300, and 600 mg/kg, respectively; orally and cisplatin 6 mg/kg intraperitonially). For the micronucleus assay, samples of blood were collected (before the first use of the drugs at T0, then 24 (T1) and 48 (T2) hours after the first administration). For the comet assay, blood samples were collected only at T2. The damage reduction percentages for the micronucleus assay were 90.0, 47.3, and 37.3% at T1 and 46.0, 38.6, and 34.7% at T2, for G6, G7, and G8 groups, respectively. For the comet assay, the damage reduction percentages were 113.0, 117.4, and 115.0% for G6, G7, and G8, respectively. We conclude that glutamine is able to prevent genotoxic and clastogenic damages caused by cisplatin.
Full-text · Article · Dec 2013 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: Chromatin is thought to modulate access of repair proteins to DNA lesions, and may be altered by chromatin remodelers to facilitate repair. We investigated the participation of chromatin remodelers and DNA repair in 5-fluorouracil (5-FU) cytotoxicity in Saccharomyces cerevisiae. 5-FU is an antineoplastic drug commonly used in clinical settings. Among the several strains tested, only those with deficiencies in ATP-dependent chromatin remodeling (CR) and some histone acetyltransferases (HAT) exhibited sensitivity to 5-FU. CR and HAT double-mutants exhibited increased resistance to 5-FU in comparison to the wild-type mutant, but were still arrested in G2/M, as were the sensitive strains. The participation of Htz1p in 5-FU toxicity was also evaluated in single- and double-mutants of CR and HAT; the most significant effect was on cell cycle distribution. 5-FU lesions are repaired by different DNA repair machineries, including homologous recombination (HR) and post-replication repair (PRR). We investigated the role of CR and HAT in these DNA repair pathways. Deficiencies in Nhp10 and CR combined with deficiencies in HR or PRR increased 5-FU sensitivity; however, combined deficiencies of HAT, HR, and PRR did not. CRs are directly recruited to DNA damage and lead to chromatin relaxation, which facilitates access of HR and PRR proteins to 5-FU lesions. Combined deficiencies in HAT with defects in HR and PRR did not potentiate 5-FU cytotoxicity, possibly because they function in a common pathway.
[Show abstract][Hide abstract] ABSTRACT: The development of new strategies for cancer therapeutics is indispensable for the improvement of standard protocols and the creation of other possibilities in cancer treatment. Yeast models have been employed to study numerous molecular aspects directly related to cancer development, as well as to determine the genetic contexts associated with anticancer drug sensitivity or resistance. The budding yeast Saccharomyces cerevisiae presents conserved cellular processes with high homology to humans, and it is a rapid, inexpensive and efficient compound screening tool. However, yeast models are still underused in cancer research and for screening of antineoplastic agents. Here, the employment of S. cerevisiae as a model system to anticancer research is discussed and exemplified. Focusing on the important determinants in genomic maintenance and cancer development, including DNA repair, cell cycle control and epigenetics, this review proposes the use of mutant yeast panels to mimic cancer phenotypes, screen and study tumor features and synthetic lethal interactions. Finally, the benefits and limitations of the yeast model are highlighted, as well as the strategies to overcome S. cerevisiae model limitations.
Full-text · Article · Aug 2012 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Adenosine diphosphate (ADP)-ribosylation is an important posttranslational modification catalyzed by a variety of enzymes,
including poly (ADP ribose) polymerases (PARPs), which use nicotinamide adenine dinucleotide (NAD+) as a substrate to synthesize and transfer ADP-ribose units to acceptor proteins. The PARP family members possess a variety
of structural domains, span a wide range of functions and localize to various cellular compartments. Among the molecular actions
attributed to PARPs, their role in the DNA damage response (DDR) has been widely documented. In particular, PARPs 1–3 are
involved in several cellular processes that respond to DNA lesions, which include DNA damage recognition, signaling and repair
as well as local transcriptional blockage, chromatin remodeling and cell death induction. However, how these enzymes are able
to participate in such numerous and diverse mechanisms in response to DNA damage is not fully understood. Herein, the DDR
functions of PARPs 1–3 and the emerging roles of poly (ADP ribose) polymers in DNA damage are reviewed. The development of
PARP inhibitors, their applications and mechanisms of action are also discussed in the context of the DDR.
[Show abstract][Hide abstract] ABSTRACT: Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. In the present study, we characterize the DNA-damage response to the antitumour acronycine derivative S23906, which forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA (replication protein A) at replication sites and rapid Chk1 activation. In contrast, neither MRN (Mre11-Rad50-Nbs1) nor ATM (ataxia-telangiectasia mutated), contributes to the initial response to S23906. Interestingly, genetic attenuation of ATR (ATM- and Ras3-related) activity inhibits not only the early phosphorylation of histone H2AX and Chk1, but also interferes with the late phosphorylation of Chk2. Moreover, loss of ATR function or pharmacological inhibition of the checkpoint kinases by AZD7762 is accompanied by abrogation of the S-phase arrest and increased sensitivity towards S23906. These findings identify ATR as a central co-ordinator of the DNA-damage response to S23906, and provide a mechanistic rationale for combinations of S23906 and similar agents with checkpoint abrogators.
[Show abstract][Hide abstract] ABSTRACT: 5-Fluorouracil (5-FU) is an antitumor antimetabolite that can be converted into fluoronucleotides and FdUMP. Fluoronucleotides are incorporated into DNA and RNA, while FdUMP results in nucleotide pool imbalance. Saccharomyces cerevisiae is unable to convert 5-FU into FdUMP, making yeast a unique model system to study the cellular effects of 5-FU and FdUMP independently. A panel of repair-deficient yeast strains was used to identify the DNA repair pathways needed for repair of lesions generated by 5-FU or FdUMP. This included yeast deficient in base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), mismatch repair (MMR), post-replication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results revealed an important role of BER, since BER-mutants (ntg1, ntg2, apn1, apn2) showed pronounced sensitivity to both 5-FU and FdUMP. MMR mutants also showed high sensitivity to both compounds. In contrast, deficiencies in NER, NHEJ and TLS repair had only minor influence on the sensitivity to FU and FdUMP. Interestingly, deficiencies in HR (rad52) and PPR (rad6, rad18) were associated with increased sensitivity to 5-FU, but not to FdUMP. Taken together, our study reveals an important contribution of DNA repair pathways on the sensitivity to 5-FU and its active metabolite FdUMP. Importantly, the repair mechanisms differed for the 2 antimetabolites since lesions induced by 5-FU were repaired by BER, MMR, HR and PRR, while only BER and MMR were required for repair of FdUMP-induced lesions.
[Show abstract][Hide abstract] ABSTRACT: This research has evaluated the effects of enteral supplementation of glutamine in clastogens and genotoxic damages caused by the acute administration of cisplatin. For this, it was utilized Swiss mice distributed in eight experimental groups: control, cisplatin, glutamine, in three different doses and the combination of these with cisplatin. The results show that the glutamine was present in neither genotoxic nor mutagenic activity. When in association with glutamine and cisplatin, in simultaneous treatment, it was verified the frequency decreased of micronuclei and comets. The damage reduction percentages to the micronucleus ranged from 95.4 to 91.8% after 24h of administration of these compounds and 76.7 to 56.8% after 48h. In the same time the damage reduction percentages to the comet test ranged from 117.0 to 115.0%. The results suggest that glutamine is capable of preventing genotoxic and mutagenic damage according to the experimental design proposed.
[Show abstract][Hide abstract] ABSTRACT: 5-Fluorouracil (5-FU) is an antineoplasic drug widely used to treat cancer. Its cytotoxic effect has been principally ascribed to the misincorporation of fluoronucleotides into DNA and RNA during their synthesis, and the inhibition of thymidylate synthase (TS) by FdUMP (one of the 5-FU active metabolites), which leads to nucleotide pool imbalance. In the present study, we compared the ability of 5-FU and FdUMP to induce apoptosis and to influence the cell cycle progression in human colon SW620 adenocarcinoma cells in regards to their genotoxic and clastogenic activities. Our study demonstrates that 5-FU induces SSB, DSB and apoptosis earlier than FdUMP. Interestingly, while both drugs are able to induce apoptosis, their effect on the cell cycle progression differed. Indeed, 5-FU induces an arrest in G1/S while FdUMP causes an arrest in G2/M. Independently of the temporal difference in strand breaks and apoptosis induction, as well as the differential cell cycle modulation, both drugs presented similar clastogenic effects. The different pattern of cell cycle arrest suggests that the two drugs induce different types of primary DNA lesions that could lead to the activation of different checkpoints and recruit different DNA repair pathways.
Full-text · Article · May 2009 · Journal of Applied Toxicology
[Show abstract][Hide abstract] ABSTRACT: The essential oil composition obtained from the leaves of Casearia sylvestris has been investigated by GC and GC/MS. Thirty-seven compounds were identified in the oil. The major constituents of which were β-caryophyllene (27.5%) and bicyclogermacrene (24.2%). The clastogenic and anticlastogenic effect of the oil was tested by chromosome aberration in cells of tissue hepatoma of Rattus novergicus. The three different concentrations of C. sylvestris oil showed clastogenic effects. However, in the tests of anticlastogenicity, the same three concentrations showed protective activity when associated with ethyl methanesulfonate (EMS).
Full-text · Article · Jul 2007 · Journal of Essential Oil Research
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT The mushroom Agaricus blazei has been extensively investigated because of evidence of its antimutagenic, antitumor, and anticarcinogenic activities. This study investigated the clastogenic and/or anticlastogenic activity of aqueous extract of Agaricus blazei (10% w/v) in drug-metabolizing rat hepatoma tissue cells (HTCs), with continuous treatment and treatment during different phases of the cell cycle. DNA damage was induced utilizing two direct-acting agents-methyl methane sulfonate and ethyl methane sulfonate-and two indirect-acting agents-2-aminoanthracene and cyclophosphamide. The aqueous extract of A. blazei with either continuous treatment or treatment during different phases of the cell cycle showed clastogenic activity. The results with continuous treatment showed that A. blazei does not protect against DNA damage-inducing agents that are direct acting. Meanwhile, when combined with indirect-acting agents, a protective effect was demonstrated. A protective effect was also found during different phases of the cell cycle when cells were treated with indirect-acting agents. The protective effects against indirect-acting agents (continuous treatment and during the different phases of the cell cycle) suggest that A. blazei may provide some health benefits to the public when used as a functional food.
Full-text · Article · Mar 2007 · Toxicology mechanisms and methods
[Show abstract][Hide abstract] ABSTRACT: A large number of functional foods, including those that contain beta-glucan, have been shown to prevent the development of cancer and other chronic diseases. The aim of the present study was to elucidate its mechanism of action, as well as to understand its effects as an antigenotoxic, anticlastogenic agent, and to determine its capacity to preserve cell viability. The investigation was carried out in the CHO-k1 and CHO-xrs5 cell lines. The cytokinesis-blocked micronucleus assay indicated that the different doses of beta-glucan examined (5, 10, 20 and 40 microg/ml) did not show clastogenic effects. In the CHO-k1 cell line, a chemopreventive effect could be observed in all the protocols tested: pre-treatment (% reduction of 35.0-57.3), simultaneous treatment (simple--5 reduction of 19.7-55.6 and with pre-incubation--of 42.7-56.4) and post-treatment (% reduction of 17.9-37.6). This finding indicates mechanisms of action involving desmutagenesis and bioantimutagenesis, albeit the latter having a lesser role. However, in the repair-deficient CHO-xrs5 cells, beta-glucan did not show a protective effect with post-treatment (% reduction of 2.96), thus supporting the involvement of bioantimutagenesis. The comet assay in CHO-k1 cells demonstrated that beta-glucan has neither a genotoxic nor an antigenotoxic effect. Cell viability tests indicated that beta-glucan preserves cell viability in both cell lines, preventing apoptotic events. These findings suggest that beta-glucan, when present in foods, could provide them with nutraceutical characteristics and act as a dietary supplement, or that beta-glucan could be used in new drug development.
Full-text · Article · Mar 2007 · Toxicology in Vitro
[Show abstract][Hide abstract] ABSTRACT: Due to the need to identify new antimutagenic agents and to determine their mechanism of action, the present study examined the mechanism of action of the beta-glucan with regard to antimutagenicity using the micronucleus assay in CHO-k1 and HTC cell lines. The mutagenicity experiments were performed with three different concentrations of beta-glucan (5, 10, and 20 microg/mL), in wich only the highest dose showed mutagenic activity. In the antimutagenicity experiments, the same concentrations of beta-glucan were combined with a mutagenic agent, methylmethane sulfonate, or 2-aminoanthracene, using four different treatment protocols: pre-treatment, simultaneous treatment (simple and with pre-incubation), and post-treatment. The results indicate that the CHO-k1 cell line treated with MMS presented a chemopreventive activity for all the doses of beta-glucan in the different treatment protocols, except for the lowest dose in post-treatment. When HTC cell line treated with MMS is analysed, a chemopreventive activity can be verified for the highest dose in both pre- and post-treatment. For the simple simultaneous treatment, the three doses demonstrated efficacy, while for the simultaneous treatment with pre-incubation only the intermediate concentration was effective. In HTC treated with 2AA both the lowest dose in the pre-treatment protocol and the post-treatment protocol did not show efficacy in preventing DNA damage. The evaluation of the different protocols and the damage decrease percentages observed suggest that beta-glucan has both desmutagenic and bioantimutagenic activity. It is necessary, however, to note that efficacy and mechanism of action are subject to variation when compared the two cell lines, since in HTC, representing a drug-metabolizing system, this substance can show a diminished chemopreventive capacity.
Full-text · Article · Nov 2006 · Toxicology in Vitro
[Show abstract][Hide abstract] ABSTRACT: The Agaricus brasiliensis Wasser & Didukh - Ab (=Agaricus blazei Murrill ss. Heinemann) is a basidiomycete that has been world wide consumed on the fight and treatment of several diseases. In this study, ovary cells (CHO-k1) in Chinese hamster , the clastogenic and genotoxic ef fects of Ab high concentrations and its protector potential, were tested, through the chromosome aberration (AC) and comet assays (SCGE), associated to two DNA repair blockers (cytosine arabinoside triphosphate - Ara- C, DNA polymerase α inhibitor and 3' deoxithymidine 5' triphosphate - 3DeoT , DNA polymerase β
[Show abstract][Hide abstract] ABSTRACT: Agaricus blazei (Ab) has become popularly known for its medicinal properties. Scientifically, it has been tested with regard to its capacity to protect genetic material against damage. We examined different organic extracts (methanolic extract -- ME, hexanic extract -- HE and n-butanolic extract -- BE) and an aqueous extract (AE) of Ab, for their capacity to induce DNA damage as well as for their protective effect. Genetic damage was determined by the chromosomal aberration assay (CA) in CHO-k1 cells for all extracts and the cytokinesis block micronucleus assay (CBMN) in non drug-metabolizing (CHO-k1) and drug-metabolizing (HTC) cell lines for extract BE only. The extracts did not show clastogenicity but showed anticlastogenicity. The greatest percent reduction obtained were with BE (105%) and AE (126%) treatments in CA. BE treatment did not display genotoxicity in CHO-k1, but was genotoxic in HTC. However, BE was shown to be antigenotoxic causing decreased micronucleus frequency in HTC and CHO-k1 cells. These results suggest that all the extracts contained protective substances, but in some cases they could show a genotoxic effect with regard to metabolism. Therefore, these findings warrant caution in the use of this mushroom by the population.
Full-text · Article · May 2006 · Toxicology in Vitro