RESUMEN
It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.
Asunto(s)
Adenocarcinoma Folicular/patología , Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Neovascularización Patológica/patología , Glándula Tiroides/patología , Neoplasias de la Tiroides/patología , Proteínas Supresoras de Tumor/metabolismo , Adenocarcinoma Folicular/irrigación sanguínea , Adenocarcinoma Folicular/metabolismo , Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Neovascularización Patológica/metabolismo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/irrigación sanguínea , Neoplasias de la Tiroides/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Reparación del ADN , Proteoma , Transcriptoma , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Animales , Antioxidantes/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , ADN Glicosilasas/genética , Endonucleasas/genética , Mutación , Purinas/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMEN
Introduction: Thyroid iodide transporters, Na+/I- symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients. Material and methods: The study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction. Results: Here we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor. Conclusions: This study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.
RESUMEN
Oxidative stress (OS) is recognized as disturbance of cellular equilibrium between reactive oxygen species (ROS) formation and their elimination by antioxidant defense systems. One example of ROS-mediated damage is generation of potentially mutagenic DNA precursor, 8-oxodGTP. In human cells genomic 8-oxodGTP incorporation is prevented by the MutT homologue 1 (MTH1 or hMTH1 for human MTH1) protein. It is well established that malignant cells, including thyroid cancer cells, require hMTH1 for maintaining proliferation and cancerous transformation phenotype. Above observations led to the development of hMTH1 inhibitors as novel anticancer therapeutics. In the current study we present extensive analysis of oxidative stress responses determining sensitivity to hMTH1 deficiency in cultured thyroid cells. We observe here that hMTH1 depletion results in downregulation of several glutathione-dependent OS defense system factors, including GPX1 and GCLM, making some of the tested thyroid cell lines highly dependent on glutathione levels. This is evidenced by the increased ROS burden and enhanced proliferation defect after combination of hMTH1 siRNA and glutathione synthesis inhibition. Moreover, due to the lack of data on hMTH1 expression in human thyroid tumor specimens we decided to perform detailed analysis of hMTH1 expression in thyroid tumor and peri-tumoral tissues from human patients. Our results allow us to propose here that anticancer activity of hMTH1 suppression may be boosted by combination with agents modulating glutathione pool, but further studies are necessary to precisely identify backgrounds susceptible to such combination treatment.
Asunto(s)
Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Regulación de la Expresión Génica , Glutatión Peroxidasa/metabolismo , Estrés Oxidativo/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Glándula Tiroides/metabolismo , Línea Celular Tumoral , Enzimas Reparadoras del ADN/genética , Glutatión Peroxidasa/genética , Humanos , Monoéster Fosfórico Hidrolasas/genética , ARN Mensajero/genética , Glándula Tiroides/citología , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Glutatión Peroxidasa GPX1RESUMEN
Cancer cells, including thyroid cancer cells, suffer from oxidative stress damaging multiple cellular targets, such as DNA and the nucleotide pool. The human MutT homologue 1 (hMTH1) controls the oxidative DNA damage load by sanitizing the nucleotide pool from the oxidized DNA precursor, 8-oxodGTP. It has previously been shown that hMTH1 is essential for cancer cell proliferation and survival, therefore hMTH1 inhibition has been proposed as a novel anticancer therapeutic strategy. Here we show that thyroid cancer cells respond to siRNA mediated hMTH1 depletion with increased DNA damage load and moderately reduced proliferation rates, but without detectable apoptosis, cell-cycle arrest or senescence. Importantly, however, hMTH1 depletion significantly reduced migration and invasion potential of the thyroid cancer cells. Accordingly, our results allow us to propose that hMTH1 may be a therapeutic target in thyroid malignancy, especially for controlling metastasis.
Asunto(s)
Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Neoplasias de la Tiroides/enzimología , Línea Celular Tumoral , Movimiento Celular , Humanos , Invasividad Neoplásica , Estrés Oxidativo , Neoplasias de la Tiroides/patologíaRESUMEN
Oxidation of DNA bases, an inevitable consequence of oxidative stress, requires the base excision repair (BER) pathway for repair. Caenorhabditis elegans is a well-established model to study phenotypic consequences and cellular responses to oxidative stress. To better understand how BER affects phenotypes associated with oxidative stress, we characterised the C. elegans nth-1 mutant, which lack the only DNA glycosylase dedicated to repair of oxidative DNA base damage, the NTH-1 DNA glycosylase. We show that nth-1 mutants have mitochondrial dysfunction characterised by lower mitochondrial DNA copy number, reduced mitochondrial membrane potential, and increased steady-state levels of reactive oxygen species. Consistently, nth-1 mutants express markers of chronic oxidative stress with high basal phosphorylation of MAP-kinases (MAPK) but further activation of MAPK in response to the superoxide generator paraquat is attenuated. Surprisingly, nth-1 mutants also failed to induce apoptosis in response to paraquat. The ability to induce apoptosis in response to paraquat was regained when basal MAPK activation was restored to wild type levels. In conclusion, the failure of nth-1 mutants to induce apoptosis in response to paraquat is not a direct effect of the DNA repair deficiency but an indirect consequence of the compensatory cellular stress response that includes MAPK activation.
Asunto(s)
Apoptosis/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ADN Glicosilasas/deficiencia , Endonucleasas/deficiencia , Células Germinativas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Caenorhabditis elegans , Respiración de la Célula , ADN Mitocondrial , Dosificación de Gen , Potencial de la Membrana Mitocondrial , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Oxidación-Reducción , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.
Asunto(s)
Proteínas Bacterianas/genética , Reparación del ADN/genética , Mutación , Animales , Proteínas Bacterianas/metabolismo , Reparación de la Incompatibilidad de ADN , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Base excision repair (BER) pathway executed by a complex network of proteins is the major system responsible for the removal of damaged DNA bases and repair of DNA single strand breaks (SSBs) generated by environmental agents, such as certain cancer therapies, or arising spontaneously during cellular metabolism. Both modified DNA bases and SSBs with ends other than 3'-OH and 5'-P are repaired either by replacement of a single or of more nucleotides in the processes called short-patch BER (SP-BER) or long-patch BER (LP-BER), respectively. In contrast to Escherichia coli cells, in human ones, the two BER sub-pathways are operated by different sets of proteins. In this review the selection between SP- and LP-BER and mutations in BER and end-processors genes and their contribution to bacterial mutagenesis and human diseases are considered.
Asunto(s)
Proteínas Bacterianas/genética , Reparación del ADN/genética , Mutación , Animales , Proteínas Bacterianas/metabolismo , Daño del ADN , Humanos , Modelos BiológicosRESUMEN
BACKGROUND: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. METHODS: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. RESULTS: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. CONCLUSIONS: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.
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Autoantígenos/análisis , Neoplasias de la Mama/patología , Mama/patología , Yoduro Peroxidasa/análisis , Proteínas de Unión a Hierro/análisis , Glándula Tiroides/patología , Autoantígenos/genética , Western Blotting , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Yoduro Peroxidasa/genética , Proteínas de Unión a Hierro/genética , Persona de Mediana EdadRESUMEN
Exocyclic adducts of DNA bases, such as etheno- and hydroxyalkano- ones, are generated by a variety of bifunctional agents, including endogenously formed products of lipid peroxidation. In this work we selectively modified cytosines in the 5'-d(TTT TTT CTT TTT CTT TTT CTT TTT T)-3' oligonucleotide using: chloroacetaldehyde to obtain 3,N(4)-alpha-hydroxyethano- (HEC) and 3,N(4)-etheno- (epsilonC), acrolein to obtain 3,N(4)-alpha-hydroxypropano- (HPC) and crotonaldehyde to obtain 3,N(4)-alpha-hydroxy-gamma-methylpropano- (mHPC) adducts of cytosine. The studied adducts are alkali-labile which results in oligonucleotide strain breaks at the sites of modification upon strong base treatment. The oligonucleotides carrying adducted cytosines were studied as substrates of Escherichia coli Mug, human TDG and fission yeast Thp1p glycosylases. All the adducts studied are excised by bacterial Mug although with various efficiency: epsilonC >HEC >HPC >mHPC. The yeast enzyme excises efficiently epsilonC>HEC>HPC, whereas the human enzyme excises only epsilonC. The pH-dependence curves of excision of eC, HEC and HPC by Mug are bell shaped and the most efficient excision of adducts occurs within the pH range of 8.6-9.6. The observed increase of excision of HEC and HPC above pH 7.2 can be explained by deprotonation of these adducts, which are high pK(a) compounds and exist in a protonated form at neutrality. On the other hand, since epsilonC is in a neutral form in the pH range studied, we postulate an involvement of an additional catalytic factor. We hypothesize that the enzyme structure undergoes a pH-induced rearrangement allowing the participation of Lys68 of Mug in catalysis via a hydrogen bond interaction of its epsilon-amino group with N(4) of the cytosine exocyclic adducts.
Asunto(s)
Disparidad de Par Base , Citosina/metabolismo , ADN Glicosilasas/metabolismo , Timina ADN Glicosilasa/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Citosina/química , Concentración de Iones de Hidrógeno , Uracil-ADN GlicosidasaRESUMEN
MutT enzymes prevent DNA damage by hydrolysis of 8-oxodGTP, an oxidized substrate for DNA synthesis and antimutagenic, anticarcinogenic, and antineurodegenerative functions of MutT enzymes are well established. MutT has been found in almost all kingdoms of life, including many bacterial species, yeasts, plants and mammals. However, a Caenorhabditis elegans MutT homologue was not previously identified. Here, we demonstrate that NDX-4 exhibits both hallmarks of a MutT-type enzyme with an ability to hydrolyze 8-oxodGTP and suppress the Escherichia coli mutT mutator phenotype. Moreover, we show that NDX-4 contributes to genomic stability in vivo in C. elegans. Phenotypic analyses of an ndx-4 mutant reveal that loss of NDX-4 leads to upregulation of key stress responsive genes that likely compensate for the in vivo role of NDX-4 in protection against deleterious consequences of oxidative stress. This discovery will enable us to use this extremely robust genetic model for further research into the contribution of oxidative DNA damage to phenotypes associated with oxidative stress.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Reparación del ADN , Inestabilidad Genómica , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Daño del ADN , Nucleótidos de Desoxiguanina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Mutación , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Monoéster Fosfórico Hidrolasas/genética , Pirofosfatasas/genética , Activación TranscripcionalRESUMEN
The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Reparación del ADN/fisiología , Mutación , Paraquat/farmacología , Uracil-ADN Glicosidasa/metabolismo , Animales , Apoptosis/efectos de la radiación , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Proteínas de Caenorhabditis elegans/genética , Daño del ADN/efectos de la radiación , Perfilación de la Expresión Génica/métodos , Uracil-ADN Glicosidasa/genéticaRESUMEN
Base excision repair (BER) is a major mode of repair of DNA base damage. BER is required for maintenance of genetic stability, which is important in the prevention of cancer. However, direct genetic associations between BER deficiency and human cancer have been difficult to firmly establish, and the first-generation mouse models deficient in individual DNA-glycosylases, which are the enzymes that give lesion specificity to the BER pathway, generally do not develop spontaneous tumors. This review summarizes our current understanding of the contribution of DNA base damage to human cancer, with a particular focus on DNA-glycosylases and two of the main enzymes that prevent misincorporation of damaged deoxynucleotide triphosphates into DNA: the dUTPase and MTH1. The available evidence suggests that the most important factors determining individual susceptibility to cancer are not mutations in individual DNA repair enzymes but rather the regulation of expression and modulation of function by protein modification and interaction partners. With this in mind, we present a comprehensive list of protein-protein interactions involving DNA-glycosylases or either of the two enzymes that limit incorporation of damaged nucleotides into DNA. Interacting partners with a known role in human cancer are specifically highlighted.
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Daño del ADN/fisiología , Reparación del ADN/fisiología , Neoplasias/genética , Animales , Secuencia de Bases , Daño del ADN/genética , Reparación del ADN/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Redes Reguladoras de Genes/fisiología , Humanos , Ratones , Modelos Biológicos , Neoplasias/metabolismo , Unión Proteica/fisiologíaRESUMEN
BACKGROUND: The level of 8-oxoguanine (8-oxoG), a general marker of oxidative DNA damage, in DNA is the result of both an equilibrium between the rates of its formation and removal from DNA by DNA repair enzymes and the removal of 8-oxodGTP from the cellular nucleotide pool by hydrolysis to 8-oxodGMP, preventing its incorporation into DNA. To determine the contribution of each component to the level of 8-oxoG in DNA, we compared 8-oxoG-excising activity (encoded by hOGG1), 8-oxodGTPase activity (encoded by hMTH1), and 8-oxoG levels in DNA from tumors and surrounding normal lung tissues from non-small-cell lung cancer patients. METHODS: We measured the level of 8-oxoG in DNA of 47 patients by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), hOGG1 activity in tissue extracts of 56 patients by the nicking assay using an oligodeoxynucleotide containing a single 8-oxoG, and hMTH1 activity in tissue extracts of 33 patients by HPLC/UV detection. All statistical tests were two-sided. RESULTS: The 8-oxoG level was lower in tumor DNA than in DNA from normal lung tissue (geometric mean: 5.81 versus 10.18 8-oxoG/10(6) G, geometric mean of difference = 1.75; P<.001). The hOGG1 activity was also lower in tumor than in normal lung tissue (geometric mean: 8.76 versus 20.91 pmol/h/mg protein, geometric mean of difference = 2.39; P<.001), whereas the hMTH1 activity was higher in tumor than in normal lung tissue (geometric mean: 28.79 versus 8.94 nmol/h/mg protein, geometric mean of difference = 0.31; P<.001). The activity of hMTH1 was three orders of magnitude higher than that of hOGG1 (nanomoles versus picomoles per hour per milligram of protein, respectively). CONCLUSIONS: Several different components contribute to the maintenance of 8-oxoG levels in human DNA, with the greatest contributor being the removal of 8-oxodGTP from the cellular nucleotide pool by hMTH1.