Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

Kaposi's sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi's sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman's disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

In vitro activation of human herpesviruses 6 and 7 from latency.

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.

Mass vaccination to control chickenpox: the influence of zoster.

The impact of transmission events from patients with shingles (zoster) on the epidemiology of varicella is examined before and after the introduction of mass immunization by using a stochastic mathematical model of transmission dynamics. Reactivation of the virus is shown to damp stochastic fluctuations and move the dynamics toward simple annual oscillations. The force of infection due to zoster cases is estimated by comparison of simulated and observed incidence time series. The presence of infectious zoster cases reduces the tendency for mass immunization to increase varicella incidence at older ages when disease severity is typically greater.

Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Reactivated and latent varicella-zoster virus in human dorsal root ganglia.

Ganglia obtained at autopsy were examined by in situ hybridization from one patient with zoster (also called herpes zoster or shingles), two varicella-zoster virus (VZV)-seropositive patients with clinical evidence of zoster, one VZV-seronegative child, and one fetus. Ganglia positive for VZV had a hybridization signal in both neuronal and nonneuronal satellite cells. Ganglia obtained from the fetus and from the seronegative infant were consistently negative for VZV. Two striking observations were evident regarding the presence of VZV DNA in ganglia obtained from the individual with zoster at the time of death. First, ganglia innervating the sites of reactivation and ganglia innervating adjacent sites yielded strongly positive signals in neurons and satellite cells, whereas ganglia from distant sites were rarely positive. Second, VZV DNA was found in both the nuclei and the cytoplasm of neurons innervating areas of zoster. However, in neurons innervating zoster-free areas, VZV DNA was found only in the nucleus of neurons and their supporting satellite cells. Immunohistochemistry with a fluorescent monoclonal antibody to the VZV glycoprotein gpI, a late virus protein, revealed a positive signal in the cytoplasm of ganglia with clinical evidence of reactivation. These results illustrate that both neuronal and satellite cells become latently infected following primary VZV infection. The presence of VZV DNA and gpI in the cytoplasm of neurons demonstrates productive infection following reactivation at the site of latency.

Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.

Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a non-V beta 8-specific superantigen.

Superantigens such as the staphylococcal enterotoxins can play an important role in exacerbation of autoimmune disorders such as experimental allergic encephalomyelitis (EAE) in mice. In fact, superantigens can reactivate EAE in PL/J mice that have been sensitized to rat myelin basic protein (MBP). The T-cell subset predominantly responsible for disease in PL/J mice bears the V beta 8+ T-cell antigen receptor (TCR). The question arises as to whether T cells bearing other V beta specificities are involved in induction or reactivation of EAE with superantigen. Thus, we have investigated the ability of a non-V beta 8-specific superantigen, staphylococcal enterotoxin A (SEA) (V beta specificities 1, 3, 10, 11, and 17), to induce EAE in PL/J mice that have been previously protected from disease by anergy and deletion of V beta 8+ T cells. PL/J mice were first pretreated with the V beta 8-specific superantigen staphylococcal enterotoxin B (SEB) and then immunized with MBP. These mice exhibited V beta 8-specific anergy and depletion and did not develop EAE, even when further treated with SEB. However, administration of SEA to these same mice induced an initial episode of EAE which was characterized by severe hindleg paralysis and accelerated onset of disease. In contrast to SEB pretreatment, PL/J mice pretreated with SEA did develop EAE when immunized with MBP, and after resolution of clinical signs of disease these mice were susceptible to relapse of EAE induced by SEB but not by SEA. Thus, superantigens can activate encephalitogenic MBP-specific non-V beta 8+ T cells to cause EAE in PL/J mice. These data suggest that superantigens can play a central role in autoimmune disorders and that they introduce a profound complexity to autoimmune diseases such as EAE, akin to the complexity seen in multiple sclerosis.

Variação sazonal do metabolismo energético no primeiro ciclo anual de lagartos teiú Tupinambis merianae: correlatos com atividades diárias, adiposidade e proteção antioxidante

Lagartos teiú eclodem no verão e enfrentam o desafio de crescer e armazenar substratos em um curto período de tempo, antes do início do período de jejum e depressão metabólica (≈80%) a temperaturas amenas durante o inverno (≈17 °C). No despertar, o aumento do metabolismo e a reperfusão de órgãos favoreceriam a ocorrência de estresse oxidativo. Na primeira parte do presente estudo investigou−se os ajustes que compatibilizam as demandas em teiús neonatos, especialmente na pré-hibernação, por meio da gravação do comportamento em vídeo e da análise da massa dos corpos gordurosos abdominais e do nível plasmático de corticosterona (CORT) durante o primeiro ciclo anual. No início do outono a massa corpórea dos teiús foi 27 g e o comprimento rostro−cloacal 9,3 cm e aumentaram 40% e 20%, respectivamente, ao longo do outono, enquanto que as taxas diminuíram progressivamente até atingirem o valor zero no início do inverno. Na primavera, a massa corpórea dos teiús aumentou 80% em relação ao despertar e dobrou em relação ao final do verão; o comprimento acumulou um aumento de 27% em relação ao final do verão. A massa relativa dos corpos gordurosos foi 3,7% no início do outono e diminuiu nos meses subsequentes; no despertar, este estoque acumulou uma perda de 63% da sua massa. No início do outono 74% dos teiús estavam ativos por 4,7 h e permaneceram 2 h assoalhando diariamente; ao longo do outono o número de animais ativos e o tempo em atividade diminuíram até que todos se tornaram inativos. Na primavera 83% dos teiús estavam ativos por 7 h e permaneceram 4 h assoalhando. Um padrão sazonal similar foi observado na atividade locomotora e na alimentação. No outono, a alimentação cessou antes da atividade diária e os teiús tornaram−se afágicos algumas semanas antes da entrada em hibernação. Os maiores níveis de CORT foram observados no início do outono, reduzindo progressivamente até valores 75 e 86% menores na dormência e despertar, respectivamente; na primavera os níveis de CORT foram 32% menores em comparação com o início do outono. Este padrão sugere um papel da CORT nos ajustes que promovem a ingestão de alimento e a deposição de substratos energéticos no outono. A redução da atividade geral no final do outono contribuiria para a economia energética e manutenção da massa corpórea, apesar da redução da ingestão de alimento. O curso temporal das alterações fisiológicas e comportamentais em neonatos reforça a ideia de que a dormência sazonal nos teiús é o resultado da expressão de um ritmo endógeno. Na segunda parte do estudo foi investigada a hipótese de que ocorreriam ajustes das defesas antioxidantes durante a hibernação, em antecipação ao despertar. Foram analisados marcadores de estresse oxidativo e antioxidantes em vários órgãos de teiús em diferentes fases do primeiro ciclo anual. A CS, um indicador do potencial oxidante, não variou no fígado e foi menor no rim e no pulmão na hibernação. As enzimas antioxidantes revelaram (1) um efeito abrangente de redução das taxas na hibernação e despertar; por exemplo, GR e CAT foram menores em todos órgãos analisados e a GST tendeu a diminuir no fígado e no rim, embora constante no coração e no pulmão. A G6PDH no fígado e no rim não variou. (2) No fígado, a GST, a Se−GPX e o teor de TBARS foram maiores na atividade de outono em relação à primavera e a Se−GPX permaneceu elevada na hibernação. (3) No fígado, a SOD foi maior na hibernação e despertar em relação ao outono e a Mn−SOD seguiu este padrão. Em contraste, no rim, coração e pulmão a SOD foi menor na hibernação e as taxas se recuperaram no coração e pulmão no despertar. A Mn−SOD seguiu este padrão no pulmão. A concentração e o estado redox da glutationa não variaram no fígado, rim e coração; no pulmão o teor de Eq−GSH e GSH foi menor na hibernação, com tendência à recuperação no despertar. O teor de PC no rim foi maior na hibernação e diminuiu no despertar. No fígado, as alterações no jejum se assemelham às sazonais, como sugerem a inibição da CAT e GR e aumento da Se−GPX. Os efeitos do jejum na primavera no rim diferem dos efeitos sazonais, como sugerem a redução do teor de Eq−GSH e GSH e o aumento da razão GSSG:GSH, a redução da G6PDH e o aumento de PC. No conjunto, houve um efeito predominante de redução das taxas enzimáticas na hibernação e no despertar, exceto pelas taxas aumentadas da SOD e Se−GPX no fígado e pela recuperação da SOD no coração e da GR, SOD e Mn−SOD no pulmão no despertar. As elevadas taxas das enzimas antioxidantes no teiú em comparação a outros ectotermos e a ausência de evidências de estresse oxidativo no despertar sugerem que a atividade enzimática remanescente é suficiente para prevenir danos aos tecidos face às flutuações do metabolismo

Macro- and microglial responses in the fellow eyes contralateral to glaucomatous eyes

Most studies employing experimental models of unilateral glaucoma have used the normotensive contralateral eye as the normal control. However, some studies have recently reported the activation of the retinal macroglia and microglia in the uninjured eye, suggesting that the eye contralateral to experimental glaucoma should not be used as a control. This review analyzes the studies describing the contralateral findings and discusses some of the routes through which the signals can reach the contralateral eye to initiate the glial reactivation.

Efeito da temperatura de revenimento sobre o grau de sensitização e resistência à corrosão por pite de aços inoxidáveis supermartensíticos contendo 13% Cr, 5% Ni, com e sem adições de Nb e Mo.

O desenvolvimento dos aços inoxidáveis Super-Martensíticos (SM) nasce da necessidade de implementar novas tecnologias, mais econômicas e amigáveis ao meio ambiente. Os aços inoxidáveis SM são uma derivação dos aços inoxidáveis martensíticos convencionais, diferenciando-se basicamente no menor teor de carbono, na adição de Ni e Mo. Foram desenvolvidos como uma alternativa para aços inoxidáveis duplex no uso de dutos para a extração de petróleo offshore em meados dos anos 90. Para que esses aços apresentem as propriedades mecânicas de resistência à tração e tenacidade é necessário que sejam realizados tratamentos de austenitização, seguido de têmpera, e de revenimento, onde, particularmente para este último, há várias opções de tempos e temperaturas. Como os tratamentos térmicos geram as propriedades mecânicas através de transformações de fase (precipitação) podem ocorrer alterações da resistência à corrosão. São conhecidos os efeitos benéficos da adição de Nb em aços inoxidáveis tradicionais. Por isso, o objetivo desta pesquisa foi estudar aços inoxidáveis SM contendo Nb. Foi pesquisada a influência da temperatura de revenimento sobre a resistência à corrosão de três aços inoxidáveis SM, os quais contêm 13% Cr, 5% Ni, 1% a 2% Mo, com e sem adições de Nb. No presente trabalho, foram denominados de SM2MoNb, SM2Mo e SM1MoNb, que representam aços com 2% Mo, 1% Mo e 0,11% Nb. Dado que os principais tipos de corrosão para aços inoxidáveis são a corrosão por pite (por cloreto) e a corrosão intergranular (sensitização), optou-se por determinar os Potenciais de Pite (Ep) e os Graus de Sensitização (GS) em função da temperatura de revenimento. Os aços passaram por recozimento a 1050°C por 48 horas, para eliminação de fase ferrita delta. Em seguida foram tratados a 1050 °C por 30 minutos, com resfriamento ao ar, para uniformização do tamanho de grão. A estrutura martensítica obtida recebeu tratamentos de revenimento em temperaturas de: 550 °C, 575 °C, 600 °C, 625 °C, 650 °C e 700 °C, por 2 horas. O GS foi medido através da técnica de reativação eletroquímica potenciodinâmica na versão ciclo duplo (DL-EPR), utilizando-se eletrólito de 1M H2SO4 + 0,01M KSCN. Para determinar o Ep foram realizados ensaios de polarização potenciodinâmica em 0,6M NaCl. Os resultados obtidos foram discutidos através das variações microestruturais encontradas. Foram empregadas técnicas de microscopia ótica (MO), microscopia eletrônica de varredura (MEV), simulação termodinâmica de fases através do programa Thermo-Calc e determinação de austenita revertida mediante difração de raios X (DRX) e ferritoscópio. A quantificação da austenita por DRX identificou que a partir de 600 °C há formação desta fase, apresentando máximo em 650 °C, e novamente diminuindo para zero a 700 °C. Por sua vez, o método do ferritoscópio detectou austenita nas condições em que a analise de DRX indicou valor nulo, sendo as mais críticas a do material temperado (sem revenimento) e do aço revenido a 700 °C. Propõe-se que tais diferenças entre os dois métodos se deve à morfologia fina da austenia retida, a qual deve estar localizada entre as agulhas de martensita. Os resultados foram discutidos em termos da precipitação de Cr23C6, Mo6C, NbC, fase Chi, austenita e ferrita, bem como das consequências do empobrecimento em Cr e Mo, gerados por tais microconstituintes. São propostos três mecanismos para explicar a sensitização: o primeiro é devido a precipitação de Cr23C6, o segundo a precipitação de fase Chi (rica em Cr e Mo) e o terceiro é devido a formação de ferrita durante o revenimento. O melhor desempenho quanto ao GS foi obtido para os revenimentos a 575 °C e 600°C, por 2 horas. Os resultados de Ep indicaram que o aço SM2MoNb, revenido a 575°C, tem o melhor desempenho quanto à resistência à corrosão por cloreto. Isso associado ao baixo GS coloca este aço, com este tratamento térmico, numa posição de destaque para aplicações onde a resistência à corrosão é um critério de seleção de material, uma vez que, segundo a literatura a temperatura de 575 °C está no intervalo de temperaturas de revenimento onde são obtidas as melhores propriedades mecânicas.