Semisynthesis of new aphidicolin derivatives with high activity against Trypanosoma cruzi.

Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.

Involvement of DNA replication in ultraviolet-induced apoptosis of mammalian cells.

Exposure of cells to ultraviolet (UV) light damages the genome and the persistence of DNA lesions triggers apoptosis in mammalian cells. RNA transcription blockage by DNA damage is believed to be implicated in signaling for UV-induced apoptosis, but the role played by DNA replication in this process is still unclear. To address this point, we have employed the DNA polymerase inhibitor aphidicolin in UV-irradiated wild-type and XPB-mutated Chinese hamster ovary cells. The data obtained with synchronized cells indicate that induction of apoptosis by UV light is independent of the cell cycle phase. Nevertheless, cells treated with aphidicolin after UV exposure showed a significant prevention of apoptosis induction when compared to proliferating cells. These results were observed in both DNA-repair proficient and deficient cells, indicating that the prevention of apoptosis by aphidicolin is independent of the cells' ability to repair the photolesions caused by UV. Taken together, these data suggest that replication of damaged DNA also leads to critical events signaling for UV-induced cell death.

P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation.

p21(Cip1/WAF1) is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UV-induced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

Localisation of aphidicolin-induced break points in Holstein-Friesian cattle (Bos taurus) using RBG-banding.

Fragile sites (FS) seem to play a role in genome instability and may be involved in karyotype evolution and chromosome aberrations. The majority of common fragile sites are induced by aphidicolin. Aphidicolin was used at two different concentrations (0.15 and 0.30 microM) to study the occurrence of FS in the cattle karyotype. In this paper, a map of aphidicolin induced break points and fragile sites in cattle chromosomes was constructed. The statistical analysis indicated that any band with three or more breaks was significantly damaged (P<0.05). According to this result, 30 of the 72 different break points observed were scored as fragile sites. The Pearson correlation test showed a positive association between chromosome length and the number of fragile sites (r=0.54). On the contrary, 21 FS were identified on negative R bands while 9 FS were located on positive R bands.

Comparative study of chromosome aberrations induced with aphidicolin in women affected by breast cancer and cervix uterine cancer.

Blood samples were obtained from 80 women: Twenty of these samples were from women affected by ductal infiltrating breast carcinoma, twenty from women affected by cervix uterine cancer, and forty individuals were screened for a control group. The search for chromosome instability that is known to affect individuals with cancer was performed through chromosome analysis in nontumor cells, intending to establish frequency and different types of numerical and structural aberrations. The results, in regard to spontaneous and aphidicolin induced chromosome aberrations, showed a significantly greater frequency (p < 0.001) of chromosome fragility, as well as other numerical and structural aberrations in breast cancer patients when compared to the control group. Similar results were obtained from cervix uterine cancer patients with the exception of certain numerical aberrations in which no significant differences were found. This suggests the existence of a certain degree of chromosomal instability affecting individuals with both types of cancer. The increase in fragility may play an important role in the biologic behavior and progression of cancer.

Ultraviolet-induced cell death is independent of DNA replication in rat kangaroo cells.

Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication.

The effects of 3-aminobenzamide and aphidicolin on x-ray-induced chromosomal aberrations in cycling and non-cycling dowwn lymphocytes

Amphidicolin (APC) e 3-aminobenzamide (3AB) foram utilizados para elucidar o papel de processos de reparo de DNA no aumento de aberraçöes induzidas por Raio-X em culturas de linfócitos de pacientes com Síndrome de Down, comparadas com controles normais. Estimulados por fitohemaglutinina (PHA) (Fase G1), os controles normais mostraram um aumento significativo na produçäo de dicêntricos, mas näo de acêntricos. Em culturas de Downs as células estimuladas mostraram um aumento significativo na produçäo de dicêntricos e acêntricos. O efeito de APC, em contraste, foi menos pronunciado sem estimulaçäo. Na condiçäo estimulado, como para 3AB, o efeito de APC foi maior em controles normais. O processamento diferencial de dados genéticos induzidos por Raio-X em linfócitos periferais de Downs, é sensível a presença de APL e 3AB. É provável que ambos, DNA polymerase Ó e poly (ADP-ribose) polimerase tem comportamento diferenciado intrínsico nos linfócitos de Downs sensíveis a PHA

Isolation and partial characterization of a high-molecular-weight DNA polymerase from Leishmania mexicana.

This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.

[Induction of fragile sites by chemical and physical agents]. / Inducción de sitios frágiles por agentes químicos y físicos.

Many human malignancies are thought to be caused in large part by environmental agents. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens appear to induce chromosomal mutations. Recently, it was suggested that chromosome aberrations (CA) might be produced by breakage at specific chromosomal points known as fragile sites (FS). FS induced by different chemical agents has been widely analyzed in lymphocytes cultures, but very little is known of the environmental factors that may influence on FS expression. No studies exist about FS expression induced with X-rays or about the interaction between radiation and chemical FS inducers. In this work FS expression induced by X-rays and 3 known FS inducers such as: BUDR, FUDR and aphidicolin, is analyzed. It was found that 17 chromosomal bands were significantly damaged (p < 0.001), thus being defined as FS. FS 3p14 and 16q23 were the more frequently observed. CA and FS levels from cultures exposed to X-rays and to FUDR plus X-rays were significantly increased (p < 0.01), suggesting that it would be convenient to use other radiosensitizer. These results suggest that FS could be induced by environmental factors such as chemical or physical agents. Finally, the correlation between FS and radiation induced CA, cancer CA and oncogene localization was established, showing the importance of the interaction between FS, CA and oncogenes in cancer development.

Induccion de sitios fragiles por agentes quimicos y fisicos / Induction of fragile sites by chemical and physical agents

En la actualidad se acepta que muchas neoplasias humanas están causadas por factores y muchos mutágenos químicos inducen aberraciones cormosómicas (AC), las caules se cree que se origina por roturas en zonas cromosómicas específicas o sitios frágiles (SF). Los SF se estudiaron en cultivos de linfocitos expuestos a diversos inductores químicos, pero todavia no se conoce como influyen los factores ambientales en su expresión. Hasta ahora no hay estudios de SF inducidos con rayos X, ni se conoce la interacción de este agente con los inductores quimicos. Este es el primer trabajo que analiza la expresión de SF inducida por rayos X y por 3 inductores de SF: BUDR, FUDR y anfidicolina. Se identificaron 17 bandas cromosómicas significtivamente afectadas (p<0.oo1), que se definieron como SF. Los SF más frecuentes estaban localizados en las bandas 3p14 y 16q23. Se observó un aumento significativo (p<0.01) de AC y SF en el cultivo expuesto a rayos X y en el tratado con FUDR más radiación, indicando la conveniencia de emplear otro agente radiosensibilizador. Los resultados observados sugerirían que muchos SF pueden ser causados por factores ambientales tales como sustancias químicas o radiación. La alta correlación establecida entre los SF y la ubicación de AC inducidos por radiación, AC en cáncer y oncogenes demostraría la importante interacción entre SF, AC y oncogenes en el proceso neoplásico

Induccion de sitios fragiles por agentes quimicos y fisicos / Induction of fragile sites by chemical and physical agents

En la actualidad se acepta que muchas neoplasias humanas están causadas por factores y muchos mutágenos químicos inducen aberraciones cormosómicas (AC), las caules se cree que se origina por roturas en zonas cromosómicas específicas o sitios frágiles (SF). Los SF se estudiaron en cultivos de linfocitos expuestos a diversos inductores químicos, pero todavia no se conoce como influyen los factores ambientales en su expresión. Hasta ahora no hay estudios de SF inducidos con rayos X, ni se conoce la interacción de este agente con los inductores quimicos. Este es el primer trabajo que analiza la expresión de SF inducida por rayos X y por 3 inductores de SF: BUDR, FUDR y anfidicolina. Se identificaron 17 bandas cromosómicas significtivamente afectadas (p<0.oo1), que se definieron como SF. Los SF más frecuentes estaban localizados en las bandas 3p14 y 16q23. Se observó un aumento significativo (p<0.01) de AC y SF en el cultivo expuesto a rayos X y en el tratado con FUDR más radiación, indicando la conveniencia de emplear otro agente radiosensibilizador. Los resultados observados sugerirían que muchos SF pueden ser causados por factores ambientales tales como sustancias químicas o radiación. La alta correlación establecida entre los SF y la ubicación de AC inducidos por radiación, AC en cáncer y oncogenes demostraría la importante interacción entre SF, AC y oncogenes en el proceso neoplásico (AU)