RESUMEN
The homologous recombination (HR) pathway has been implicated as the predominant mechanism for the repair of chromosomal DNA double-strand breaks (DSBs) of the malarial parasite. Although the extrachromosomal mitochondrial genome of this parasite experiences a greater number of DSBs due to its close proximity to the electron transport chain, nothing is known about the proteins involved in the repair of the mitochondrial genome. We investigated the involvement of nucleus-encoded HR proteins in the repair of the mitochondrial genome, as this genome does not code for any DNA repair proteins. Here, we provide evidence that the nucleus-encoded "recombinosome" of the parasite is also involved in mitochondrial genome repair. First, two crucial HR proteins, namely, Plasmodium falciparum Rad51 (PfRad51) and P. falciparum Bloom helicase (PfBlm) are located in the mitochondria. They are recruited to the mitochondrial genome at the schizont stage, a stage that is prone to DSBs due to exposure to various endogenous and physiologic DNA-damaging agents. Second, the recruitment of these two proteins to the damaged mitochondrial genome coincides with the DNA repair kinetics. Moreover, both the proteins exit the mitochondrial DNA (mtDNA) once the genome is repaired. Most importantly, the specific chemical inhibitors of PfRad51 and PfBlm block the repair of UV-induced DSBs of the mitochondrial genome. Additionally, overexpression of these two proteins resulted in a kinetically faster repair. Given the essentiality of the mitochondrial genome, blocking its repair by inhibiting the HR pathway could offer a novel strategy for curbing malaria. IMPORTANCE The impact of malaria on global public health and the world economy continues to surge despite decades of vaccine research and drug development efforts. An alarming rise in resistance toward all the commercially available antimalarial drugs and the lack of an effective malaria vaccine brings us to the urge to identify novel intervention strategies for curbing malaria. Here, we uncover the molecular mechanism behind the repair of the most deleterious form of DNA lesions on the parasitic mitochondrial genome. Given that the single-copy mitochondrion is an indispensable organelle of the malaria parasite, we propose that targeting the mitochondrial DNA repair pathways should be exploited as a potential malaria control strategy. The establishment of the parasitic homologous recombination machinery as the predominant repair mechanism of the mitochondrial DNA double-strand breaks underscores the importance of this pathway as a novel druggable target.
Asunto(s)
Antimaláricos/farmacología , Genoma Mitocondrial/efectos de los fármacos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , RecQ Helicasas/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Recombinación Homóloga , Humanos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
As the energy factory for the cell, the mitochondrion, through its role of adenosine triphosphate production by oxidative phosphorylation, can be regarded as the guardian of well regulated cellular metabolism; the integrity of mitochondrial functions, however, is particularly vulnerable in cancer due to the lack of superstructures such as histone and lamina folds to protect the mitochondrial genome from unintended exposure, which consequently elevates risks of mutation. In cancer, mechanisms responsible for enforcing quality control surveillance for identifying and eliminating defective mitochondria are often poorly regulated, and certain uneliminated mitochondrial DNA (mtDNA) mutations and polymorphisms can be advantageous for the proliferation, progression, and metastasis of tumor cells. Such pathogenic mtDNA aberrations are likely to increase and occasionally be homoplasmic in cancer cells and, intriguingly, in normal cells in the proximity of tumor microenvironments as well. Distinct characteristics of these abnormalities in mtDNA may provide a new path for cancer therapy. Here we discuss a promising novel therapeutic strategy, using the sequence-specific properties of pyrrole-imidazole polyamide-triphenylphosphonium conjugates, against cancer for clearing abnormal mtDNA by reactivating mitochondrial quality control surveillance.
Asunto(s)
Mitocondrias/genética , Neoplasias/genética , Compuestos Organofosforados/farmacología , Genoma Mitocondrial/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Terapia Molecular Dirigida , Mutación , Neoplasias/tratamiento farmacológico , Compuestos Organofosforados/química , Compuestos Organofosforados/uso terapéuticoRESUMEN
Lead is a public health hazard substance affecting millions of people worldwide especially those who are occupationally exposed. Our study aimed to investigate the effect of occupational lead exposure on mitochondria DNA (mtDNA). By sequencing the whole mitochondria genome, we identified 25 unique variants in lead exposed subjects affecting 10 protein coding genes in the order of MT-ND1, MT-ND2, MT-CO2, MT-ATP8, MT-ATP6, MT-CO3, MT-ND3, MT-ND4, MT-ND5, and MT-CYB. Mitochondria functional analysis revealed that exposure to lead can reduce reactive oxygen species (ROS) levels, alter mitochondria membrane potential (MMP) and increase mitochondrial mass (MM). This was further supported by mtDNA copy number analysis which was increased in lead exposed individuals compared to unexposed control group indicating the compensatory mechanism that lead has in stabilizing the mitochondria. This is the first report of mtDNA mutation and copy number analysis in occupationally lead exposed subjects where we identified mtDNA mutation signature associated with lead exposure thus providing evidence for altered molecular mechanism to compensate mitochondrial oxidative stress.
Asunto(s)
Genoma Mitocondrial/efectos de los fármacos , Genoma Mitocondrial/genética , Plomo/efectos adversos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mutación/efectos de los fármacos , Mutación/genética , Adulto , ADN Mitocondrial/genética , Genes Mitocondriales/efectos de los fármacos , Genes Mitocondriales/genética , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The present study investigated the vitrification-induced deterioration of mitochondrial functions that may reduce the developmental ability of post-warming bovine embryos. In addition, the effect of supplementation of the culture medium with resveratrol on the mitochondrial functions and post-warming embryonic development was examined. Two days after in vitro fertilization, embryos with 8-12 cells (referred to hereafter as 8-cell embryos) were vitrified and warmed, followed by in vitro incubation for 5 days in a culture medium containing either the vehicle or 0.5 µM resveratrol. Vitrification reduced embryonic development until the blastocyst stage, reduced the ATP content of embryos, and impaired the mitochondrial genome integrity, as determined by real-time polymerase chain reaction. Although the total cell number and mitochondrial DNA copy number (Mt-number) of blastocysts were low in the vitrified embryos, the Mt-number per blastomere was similar among the blastocysts derived from fresh (non-vitrified) and vitrified-warmed embryos. Supplementation of the culture medium with resveratrol enhanced the post-warming embryonic development and reduced the Mt-number and reactive oxygen species level in blastocysts and blastomeres without affecting the ATP content. An increase in the content of cell-free mitochondrial DNA in the spent culture medium was observed following cultivation of embryos with resveratrol. These results suggested that vitrification induces mitochondrial damages and that resveratrol may enhance the development of post-warming embryos and activates the degeneration of damaged mitochondria, as indicated by the increase in the cell-free mitochondrial DNA content in the spent culture medium and the decrease in the Mt-number of blastocysts and blastomeres.
Asunto(s)
Criopreservación , Crioprotectores/farmacología , Desarrollo Embrionario/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Resveratrol/farmacología , Vitrificación , Adenosina Trifosfato/metabolismo , Animales , Blastómeros/efectos de los fármacos , Blastómeros/metabolismo , Bovinos , Criopreservación/métodos , Variaciones en el Número de Copia de ADN/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , Genoma Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Técnicas Reproductivas Asistidas , Vitrificación/efectos de los fármacosRESUMEN
Modelling of pathological processes in cells is one of the most sought-after technologies of the 21st century. Using models of such processes may help to study the pathogenetic mechanisms of various diseases. The aim of the present study was to analyse the literature, dedicated to obtaining and investigating cybrid models. Besides, the possibility of modeling pathological processes in cells and treatment of different diseases using the models was evaluated. Methods of obtaining Rho0 cell cultures showed that, during their creation, mainly a standard technique, based on the use of mtDNA replication inhibitors (ethidium bromide), was applied. Cybrid lines were usually obtained by PEG fusion. Most frequently, platelets acted as donors of mitochondria. According to the analysis of the literature data, cybrid cell cultures can be modeled to study the dysfunction of the mitochondrial genome and molecular cellular pathological processes. Such models can be very promising for the development of therapeutic approaches to the treatment of various human diseases.
Asunto(s)
ADN Mitocondrial/genética , Genoma Mitocondrial/genética , Animales , ADN Mitocondrial/efectos de los fármacos , Etidio/farmacología , Genoma Mitocondrial/efectos de los fármacos , Células HEK293 , Humanos , Mutación/efectos de los fármacos , Mutación/genéticaRESUMEN
Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of Trypanosoma brucei on its mitochondrial genome (kinetoplast DNA [kDNA]), a target of the antitrypanosomatid drug isometamidium. Here, we report that V-ATPase inhibition with bafilomycin A1 (BafA) provides partial resistance to genetic knockdown of mitochondrial gene expression. BafA does not promote long-term survival after kDNA loss, but in its presence, isometamidium causes less damage to kDNA.
Asunto(s)
Genes Mitocondriales/efectos de los fármacos , Genoma Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , ADN de Cinetoplasto/efectos de los fármacos , ADN de Cinetoplasto/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Técnicas de Silenciamiento del Gen/métodos , Genes Mitocondriales/genética , Genoma Mitocondrial/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Fenantridinas/farmacología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
There is growing evidence that the sequence variation of mitochondrial DNA (mtDNA), which clusters in population- and/or geographic-specific haplogroups, may result in functional effects that, in turn, become relevant in disease predisposition or protection, interaction with environmental factors and ultimately in modulating longevity. To unravel functional differences between mtDNA haplogroups we here employed transmitochondrial cytoplasmic hybrid cells (cybrids) grown in galactose medium, a culture condition that forces oxidative phosphorylation, and in the presence of rotenone, the classic inhibitor of respiratory Complex I. Under this experimental paradigm we assessed functional parameters such as cell viability and respiration, ATP synthesis, reactive oxygen species production and mtDNA copy number. Our analyses show that haplogroup J1, which is common in western Eurasian populations, is the most sensitive to rotenone, whereas K1 mitogenomes orchestrate the best compensation, possibly because of the haplogroup-specific missense variants impinging on Complex I function. Remarkably, haplogroups J1 and K1 fit the genetic associations previously established with Leber's hereditary optic neuropathy (LHON) for J1, as a penetrance enhancer, and with Parkinson's disease (PD) for K1, as a protective background. Our findings provide functional evidences supporting previous well-established genetic associations of specific haplogroups with two neurodegenerative pathologies, LHON and PD. Our experimental paradigm is instrumental to highlighting the subtle functional differences characterizing mtDNA haplogroups, which will be increasingly needed to dissect the role of mtDNA genetic variation in health, disease and longevity.
Asunto(s)
ADN Mitocondrial/genética , Genoma Mitocondrial/genética , Haplotipos/genética , Enfermedad de Parkinson Secundaria/genética , Plaguicidas/toxicidad , Rotenona/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , ADN Mitocondrial/química , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Genoma Mitocondrial/efectos de los fármacos , Haplotipos/efectos de los fármacos , Humanos , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Enfermedad de Parkinson Secundaria/inducido químicamente , Filogenia , Estructura Secundaria de ProteínaRESUMEN
MMS19 localizes to the cytoplasmic and nuclear compartments involved in transcription and nucleotide excision repair (NER). However, whether MMS19 localizes to mitochondria, where it plays a role in maintaining mitochondrial genome stability, remains unknown. In this study, we provide the first evidence that MMS19 is localized in the inner membrane of mitochondria and participates in mtDNA oxidative damage repair. MMS19 knockdown led to mitochondrial dysfunctions including decreased mtDNA copy number, diminished mtDNA repair capacity, and elevated levels of mtDNA common deletion after oxidative stress. Immunoprecipitation - mass spectrometry analysis identified that MMS19 interacts with ANT2, a protein associated with mitochondrial ATP metabolism. ANT2 knockdown also resulted in a decreased mtDNA repair capacity after oxidative damage. Our findings suggest that MMS19 plays an essential role in maintaining mitochondrial genome stability.
Asunto(s)
ADN Mitocondrial/metabolismo , Genoma Mitocondrial/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Estrés Oxidativo , Factores de Transcripción/metabolismo , Células Cultivadas , ADN Mitocondrial/efectos de los fármacos , Genoma Mitocondrial/efectos de los fármacos , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Mitochondria are important cellular organelles that function as control centers of the energy supply for highly proliferative cancer cells and regulate apoptosis after cancer chemotherapy. Cisplatin is one of the most important chemotherapeutic agents and a key drug in therapeutic regimens for a broad range of solid tumors. Cisplatin may directly interact with mitochondria, which can induce apoptosis. The direct interactions between cisplatin and mitochondria may account for our understanding of the clinical activity of cisplatin and development of resistance. However, the basis for the roles of mitochondria under treatment with chemotherapy is poorly understood. In this review, we present novel aspects regarding the unique characteristics of the mitochondrial genome in relation to the use of platinum-based chemotherapy and describe our recent work demonstrating the importance of the mitochondrial transcription factor A (mtTFA) expression in cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Genoma Mitocondrial/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Neoplasias/tratamiento farmacológico , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Daño del ADN , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Neoplasias/metabolismo , Estrés OxidativoRESUMEN
Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM2.5 exposure was calculated for each participant's home address using a dispersion model. An interquartile range (IQR) increment in PM2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of -15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Mitocondrias/genética , Placenta/efectos de los fármacos , ARN Ribosómico/genética , Metilación de ADN/genética , Exposición a Riesgos Ambientales , Epigénesis Genética , Femenino , Genoma Mitocondrial/efectos de los fármacos , Humanos , Recién Nacido , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Estrés Oxidativo/efectos de los fármacos , Material Particulado/toxicidad , Placenta/patología , EmbarazoRESUMEN
Cassia angustifolia Vahl. (senna) is commonly used in self-medication and is frequently used to treat intestine constipation. A previous study involving bacteria and plasmid DNA suggested the possible toxicity of the aqueous extract of senna (SAE). The aim of this study was to extend the knowledge concerning SAE genotoxicity mechanisms because of its widespread use and its risks to human health. We investigated the impact of SAE on nuclear DNA and on the stability of mitochondrial DNA in Saccharomyces cerevisiae (wt, ogg1, msh6, and ogg1msh6) strains, monitoring the formation of petite mutants. Our results demonstrated that SAE specifically increased Can(R) mutagenesis only in the msh6 mutant, supporting the view that SAE can induce misincorporation errors in DNA. We observed a significant increase in the frequency of petite colonies in all studied strains. Our data indicate that SAE has genotoxic activity towards both mitochondrial and nuclear DNA.
Asunto(s)
Núcleo Celular/genética , Genoma Mitocondrial/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Extractos Vegetales/farmacología , Saccharomyces cerevisiae/genética , Senna/química , Agua/química , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Deletions and mutations in mitochondrial DNA (mtDNA), which are frequent in human tumors, such as hepatocellular carcinoma (HCC), may contribute to enhancing their malignant phenotype. Here we have investigated the effect of mtDNA depletion in the expression of genes accounting for mechanisms of chemoresistance (MOC) in HCC. Using human HCC SK-Hep-1 cells depleted of mtDNA (Rho), changes in gene expression in response to antitumor drugs previously assayed in HCC treatment were analyzed. In Rho cells, a decreased sensitivity to doxorubicin-, SN-38-, cisplatin (CDDP)-, and sorafenib-induced cell death was found. Both constitutive and drug-induced reactive oxygen species generation were decreased. Owing to activation of the NRF2-mediated pathway, MDR1, MRP1, and MRP2 expression was higher in Rho than in wild-type cells. This difference was maintained after further upregulation induced by treatment with doxorubicin, SN-38, or CDDP. Topoisomerase-IIa expression was also enhanced in Rho cells before and after treatment with these drugs. Moreover, the ability of doxorubicin, SN-38 and CDDP to induce proapoptotic signals was weaker in Rho cells, as evidenced by survivin upregulation and reductions in Bax/Bcl-2 expression ratios. Changes in these genes seem to play a minor role in the enhanced resistance of Rho cells to sorafenib, which may be related to an enhanced intracellular ATP content together with the loss of expression of the specific target of sorafenib, tyrosine kinase receptor Kit. In conclusion, these results suggest that mtDNA depletion may activate MOC able to hinder the efficacy of chemotherapy against HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Resistencia a Antineoplásicos/genética , Expresión Génica/genética , Genoma Mitocondrial/genética , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Genoma Mitocondrial/efectos de los fármacos , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Exposure to arsenic (As) causes serious health hazards. Therefore, there is a sustained effort to understand the molecular basis of the risk posed by the toxicant. It has been reported that apoptotic changes ensue on exposure to As. To investigate the molecular basis of such changes, we sequenced the entire mitochondrial (mt) genome from PBMC of a subset of these individuals (As-exposed=16 and unexposed=18) using Affymetrix platform. Our analysis revealed that As exposure does not induce large-scale mt-DNA variations, but that specific deleterious changes could induce mt dysfunction. A Glu115Ter mutation as well as 17 other in silico predicted deleterious variants were identified exclusively in exposed individuals. The number of variants in mt Complex I in As-exposed individuals was positively correlated with their respective intracellular ROS level. In addition, the extent of potentially damaging variants in As-exposed individuals had significant positive correlation to the degree of G0 /G1 cell cycle arrest.
Asunto(s)
Apoptosis/genética , Arsénico/toxicidad , Variación Genética , Genoma Mitocondrial/efectos de los fármacos , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Humanos , Mutación , Especies Reactivas de Oxígeno/sangreRESUMEN
Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.
Asunto(s)
ADN de Cinetoplasto/efectos de los fármacos , Etidio/farmacología , Trypanosoma/efectos de los fármacos , Antiprotozoarios/farmacología , Replicación del ADN/efectos de los fármacos , ADN de Forma Z , Genoma Mitocondrial/efectos de los fármacos , Conformación de Ácido Nucleico , Trypanosoma brucei brucei , Tripanosomiasis AfricanaRESUMEN
The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.
Asunto(s)
Caenorhabditis elegans/genética , Genes Mitocondriales , Genoma Mitocondrial/fisiología , Mitocondrias/metabolismo , Modelos Animales , Animales , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , ADN Polimerasa gamma , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/fisiología , Dosificación de Gen/genética , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genoma Mitocondrial/efectos de los fármacos , Genoma Mitocondrial/genética , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Inhibidores de la Síntesis del Ácido Nucleico , Organismos Modificados Genéticamente , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacologíaRESUMEN
OBJECTIVES: To investigate somatic mitochondrial DNA mutation in primary and recurrent ovarian carcinoma tissues as well as that in drug-resistant cell lines to illuminate the impact of chemotherapeutic drugs on mitochondrial DNA (mtDNA). METHODS: Complete mtDNA genomes of 20 pairs of ovarian carcinomas and their matched normal tissues together with 2 ovarian carcinoma cell lines and their 4 platinum-resistant cell lines were sequenced. Mitochondrial DNA alterations, consequent amino acid alterations were compared between the 2 groups of patients and the 2 types of cell lines. RESULTS: A large number of mtDNA new polymorphisms (55) and mutations (18) were identified in 20 ovarian carcinoma samples. Platinum-based chemotherapy did not increase the number of new polymorphisms (P = 0.094), mutations (P = 0.688), and consequent amino acid alterations (P = 0.202 and 0.795). Data gained from the cell lines also indicated that platinum had some effect on the mitochondrial genome but not specific to particular positions. CONCLUSIONS: What we found suggested that mtDNA damage could be made by chemotherapeutic drugs but not as much as imagined in ovarian carcinomas. Some of the mtDNA defects might be part of the disease processes and cell properties as well as a consequence of treatment.
Asunto(s)
ADN Mitocondrial/genética , Genoma Mitocondrial/efectos de los fármacos , Compuestos Organoplatinos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/secundario , Adulto , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/secundario , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/secundario , Femenino , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Mutación/genética , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genética , Células Tumorales CultivadasRESUMEN
The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.
Asunto(s)
Daño del ADN , ADN Mitocondrial/efectos de los fármacos , Epiclorhidrina/toxicidad , Compuestos Epoxi/toxicidad , Alquilantes/toxicidad , Animales , Apoptosis , Secuencia de Bases , Línea Celular , Núcleo Celular/efectos de los fármacos , Pollos , Reactivos de Enlaces Cruzados/toxicidad , ADN/efectos de los fármacos , ADN/genética , Cartilla de ADN/genética , Reparación del ADN , ADN Mitocondrial/genética , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Genoma Mitocondrial/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
The nucleotide sequence of the complete mitochondrial DNA (mtDNA) molecule of the salt-water crocodile (Crocodylus porosus) was determined in this article. The molecule is 16,917 base pairs (bp) in length, and codes for 22 tRNAs, 13 protein-coding genes, 2 rRNAs, as well as a control region (D-loop), as is characteristic for mitochondrial genomes of other metazoans. The gene order conforms to that of other crocodilians sequenced, but the arrangement of some tRNA genes differs from other vertebrates. It shows that the gene order of crocodilians is remarkably conserved. In this study, the relationships among crocodilians were examined in the phylogenetic analysis based on the control conserved regions of 17 crocodilians. The results suggest that the gharial (Gavialis gangeticus) joins the false gharial (Tomistoma schlegelii) on a common branch, and then constitutes a sister group to traditional Crocodylidae. Thus, the result supports that G. gangeticus belongs to Crocodylidae. The analyses also suggest that the African slender-snouted crocodile (Crocodylus cataphractus) can be treated as an isolated genus, and constitutes a sister group to Crocodylus.