The new great promise in drug innovation: RNA interference from the laboratory to the clinic

The discovery of gene silencing mechanisms in our own cells using RNA interference is very recent. However, in less than a decade, the scientific investigation have progressed enough to make us see that, very soon, we will use this knowledge for therapeutic purposes. RNA duplexes are potential pharmaceutical drugs and there are high investments in this new strategy. The promising gene therapy seems to finally reach maturity with these new tools.A descoberta de que nossas células dispõem de um mecanismo de silenciamento gênico empregando RNA interferência ainda é muito recente. Apesar disso, em menos de uma década a investigação científica já alcançou progresso, suficiente para muito brevemente nos apropriarmos desse conhecimento com fins terapêuticos. Duplexes de RNA são potenciais fármacos e há investimentos altos nessa nova abordagem. Aparentemente, a promessa de terapia gênica parece finalmente atingir sua maturidade com essas novas ferramentas

UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts

DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.FAPESPCNP

The first of a series of articles dedicated to the 60th anniversary of the Brazilian Society of Genetics (SBG)

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The third of a series of article for the 60th anniversary of the brazilian society of genetics

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DNA repair pathways and cisplatin resistance: an intimate relationship

The main goal of chemotherapeutic drugs is to induce massive cell death in tumors. Cisplatin is an antitumor drug widely used to treat several types of cancer. Despite its remarkable efficiency, most tumors show intrinsic or acquired drug resistance. The primary biological target of cisplatin is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. These cisplatin-induced DNA lesions strongly induce cell death if they are not properly repaired or processed. To counteract cisplatin-induced DNA damage, cells use an intricate network of mechanisms, including DNA damage repair and translesion synthesis. In this review, we describe how cisplatin-induced DNA lesions are repaired or tolerated by cells and focus on the pivotal role of DNA repair and tolerance mechanisms in tumor resistance to cisplatin. In fact, several recent clinical findings have correlated the tumor cell status of DNA repair/translesion synthesis with patient response to cisplatin treatment. Furthermore, these mechanisms provide interesting targets for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy

ATM Pathway Is Essential for HPV-Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation.

Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors

El extracto acuoso de Cymbopogon citratus protege al ADN plasmídico del daño inducido por radiación UVC

Aim: to evaluate the photoprotective effect of aqueous extract of Cymbopogon citratus (DC) Stapf against UVC-induced damage to ADN. Material and methods: In the experimental procedure, samples of plasmid pBluescript SK II solutions were exposed to C. citratus aqueous extract in 0.01-4.0 mg/mL concentrations during 30, 60 and 90 min. In order to evaluate the photoprotective effect, the vegetal extract was applied before, during and after UVC radiation at 200 J/m2 doses. DNA repair enzymes T4 endonuclease V was employed in order to discriminate CPDs damage. Then, supercoiled and relaxed forms of DNA were separated after electrophoretic migration in agarose gels. Also aqueous extract transmittance was measure at 254 nm OD. Results: None of the concentrations tested were genotoxic in 30 min of exposition. Concentrations ≥ 2 mg/mL induced strand breaks at 90 min of incubation. The C. citratus extract at concentrations ≥ 0.5 mg/mL protect DNA in front of UVC radiation. Conclusions: In our experimental conditions, C. citratus extract protects DNA from the genotoxicity induced by light UVC, preventing the CPDs generation, but is not able to eliminate DNA damage once formed.Objetivo: Evaluar el efecto protector del extracto acuoso de Cymbopogon citratus (DC) Stapf, ante el daño inducido por las radiaciones UVC. Material y Métodos: Para evaluar si el extracto acuoso de C. citratus era capaz de inducir roturas de cadenas en el ADN, moléculas de plásmido pBluescript SK II fueron tratadas con diferentes concentraciones del extracto (0,01 - 4,0 mg/mL), en los tiempos de exposición: 30, 60 y 90 min. El efecto fotoprotector fue evaluado aplicando el extracto vegetal antes, durante, y después de la irradiación del ADN plasmídico con 200 J/m2 de UVC. La actividad enzimática de T4 endonucleasa V fue empleada para detectar formación de CPDs. Las formas superenrollada y relajada de las moléculas de plásmido fueron separadas electroforéticamente en gel de agarosa. Adicionalmente, se midió la transmitancia del extracto acuoso a la DO de 254 nm. Resultados: Ninguna de las concentraciones evaluadas resultó genotóxica con 30 min de tratamiento. Las concentraciones ≥ 2 mg/mL indujeron roturas de cadenas a los 90 min de incubación. El extracto de C. citratus a concentraciones ≥ 0,5 mg/mL protegió al ADN frente a las radiaciones UVC. Conclusiones: En nuestras condiciones experimentales, el extracto acuoso de C. citratus protege al ADN frente a la genotoxicidad inducida por la luz UVC, previniendo la generación de CPDs, pero no es capaz de eliminarlas una vez formadas

Cymbopogon citratus aqueous extract protects plasmid DNA from UVC-induced damage

Esta investigación fue financiada por un proyecto de colaboración bilateral entre Brasil y Cuba, CAPES/MES.Objetivo: Evaluar el efecto protector del extracto acuoso de Cymbopogon citratus (DC) Stapf, ante el daño inducido por las radiaciones UVC. Material y Métodos: Para evaluar si el extracto acuoso de C. citratus era capaz de inducir roturas de cadenas en el ADN, moléculas de plásmido pBluescript SK II fueron tratadas con diferentes concentraciones del extracto (0,01 - 4,0 mg/mL), en los tiempos de exposición: 30, 60 y 90 min. El efecto fotoprotector fue evaluado aplicando el extracto vegetal antes, durante, y después de la irradiación del ADN plasmídico con 200 J/m2 de UVC. La actividad enzimática de T4 endonucleasa V fue empleada para detectar formación de CPDs. Las formas superenrollada y relajada de las moléculas de plásmido fueron separadas electroforéticamente en gel de agarosa. Adicionalmente, se midió la transmitancia del extracto acuoso a la DO de 254 nm. Resultados: Ninguna de las concentraciones evaluadas resultó genotóxica con 30 min de tratamiento. Las concentraciones ≥ 2 mg/mL indujeron roturas de cadenas a los 90 min de incubación. El extracto de C. citratus a concentraciones ≥ 0,5 mg/mL protegió al ADN frente a las radiaciones UVC. Conclusiones: En nuestras condiciones experimentales, el extracto acuoso de C. citratus protege al ADN frente a la genotoxicidad inducida por la luz UVC, previniendo la generación de CPDs, pero no es capaz de eliminarlas una vez formadas.Aim: to evaluate the photoprotective effect of aqueous extract of Cymbopogon citratus (DC) Stapf against UVC-induced damage to ADN. Material and methods: In the experimental procedure, samples of plasmid pBluescript SK II solutions were exposed to C. citratus aqueous extract in 0.01-4.0 mg/mL concentrations during 30, 60 and 90 min. In order to evaluate the photoprotective effect, the vegetal extract was applied before, during and after UVC radiation at 200 J/m2 doses. DNA repair enzymes T4 endonuclease V was employed in order to discriminate CPDs damage. Then, supercoiled and relaxed forms of DNA were separated after electrophoretic migration in agarose gels. Also aqueous extract transmittance was measure at 254 nm OD. Results: None of the concentrations tested were genotoxic in 30 min of exposition. Concentrations ≥ 2 mg/mL induced strand breaks at 90 min of incubation. The C. citratus extract at concentrations ≥ 0.5 mg/ mL protect DNA in front of UVC radiation. Conclusions: In our experimental conditions, C. citratus extract protects DNA from the genotoxicity induced by light UVC, preventing the CPDs generation, but is not able to eliminate DNA damage once formed.Este trabajo fue realizado por el proyecto de colaboración internacional CAPES (Brasil)- MES (Cuba). El financiamiento y soporte fue brindado por CAPES (São Paulo, Brazil)

Sunlight damage to cellular DNA : focus on oxidatively generated lesions.

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells