The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism

The SOS screen, as originally described by Perkins et al. (1999), was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS+ candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS+ candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS+ candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Inhibition of replication of rinderpest virus by 5-fluorouracil

5-Fluorouracil (5FU), an analogue of uracil, was found to inhibit the production of infectious particles of rinderpest virus (RPV) in Vero cells (African green monkey kidney cells) by 99%, at a concentration of 1 μg/ml. The levels of individual mRNA specific for five of the virus genes were also reduced drastically, while the level of mRNA for a cellular housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was unaltered by fluorouracil treatment of infected cells. Both virus RNA and protein synthesis showed inhibition in a dose-dependent manner. The virions which budded out of 5-fluorouracil-treated cells also contained reduced amounts of virus proteins compared with virus particles from untreated cells.

Estudio de la trabeculectomía experimental mediante implante de PLGA como dispositivo de liberación controlada de fármacos

225 p. : il.

Incorporation of novel 1-alkylcarbonyloxymethyl prodrugs of 5-fluorouracil into poly(lactide-co-glycolide) nanoparticles

Incorporation of 1-alkylcarbonyloxymethylprodrugs of 5FU into poly(lactide-co-glycolide) nanoparticles using nanoprecipitation methods gave increased loading efficiencies over that obtained using the parent drug substance. SEM studies revealed spherical nanoparticles of around 200 nm in diameter, corresponding well with measurements made using photon correlation spectroscopy. The C-7 prodrug gave the best mean loading of 47.23%, which compared favourably to 3.68% loading achieved with 5FU. Loading efficiency was seen to follow the hydrophilic-lipophilic balance in the homologue series, where increases in lipophilicities alone were not good predictors of loading. Drug release, in terms of resultant 5FU concentration, was monitored using a flow-through dissolution apparatus. Cumulative drug release from nanoparticles loaded with the C-5 prodrug was linear over 6h, with approximately 14% of the total available 5FU dose released and with no evidence of a burst effect. The flux profile of the C-5-loaded nanoparticles showed an initial peak in flux in the first sampling interval, but became linear for the remainder of the release phase. C-7-loaded nanoparticles released considerably less (4% in 6 h) with a similar flux pattern to that seen with the C-5 prodrug. The C-9-loaded nanoparticles released less than 1% of the available 5FU over 6 h, with a similar zero-order profile. The C7 prodrug was deemed to be the prodrug of choice, achieving the highest loadings and releasing 5FU, following hydrolysis, in a zero-order fashion over a period of at least 6 h. Given the lack of burst effect and steady-state flux conditions, this nanoparticulate formulation offers a better dosing strategy for sustained intravenous use when compared to that arising from nanoparticles made by direct incorporation of 5FU. (c) 2007 Elsevier B.V. All rights reserved.

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

Oxaliplatin/capecitabine versus oxaliplatin/infusional 5-FU in advanced colorectal cancer: The MRC COIN Trial.

Background:

COIN compared first-line continuous chemotherapy with the same chemotherapy given intermittently or with cetuximab in advanced colorectal cancer (aCRC).


Methods:

Choice between oxaliplatin/capecitabine (OxCap) and oxaliplatin/leucovorin (LV)/infusional 5-FU (OxFU) was by physician and patient choice and switching regimen was allowed. We compared OxCap with OxFU and OxCap+cetuximab with OxFU+cetuximab retrospectively in patients and examined efficacy, toxicity profiles and the effect of mild renal impairment.


Results:

In total, 64% of 2397 patients received OxCap(±cetuximab). Overall survival, progression free survival and overall response rate were similar between OxCap and OxFU but rate of radical surgeries was higher for OxFU. Progression free survival was longer for OxFU+cetuximab compared with OxCap+cetuximab but other efficacy measures were similar. Oxaliplatin/LV/infusional 5-FU (±cetuximab) was associated with more mucositis and infection whereas OxCap(±cetuximab) caused more gastrointestinal toxicities and palmar-plantar erythema. In total, 118 patients switched regimen, mainly due to toxicity; only 16% came off their second regimen due to intolerance. Patients with creatinine clearance (CrCl) 50–80?ml?min-1 on OxCap(±cetuximab) or OxFU+cetuximab had more dose modifications than those with better renal function.


Conclusions:

Overall, OxFU and OxCap are equally effective in treating aCRC. However, the toxicity profiles differ and switching from one regimen to the other for poor tolerance is a reasonable option. Patients with CrCl 50–80?ml?min-1 on both regimens require close toxicity monitoring.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.

Molecular pathology of colorectal cancer.

Colorectal cancer (CRC) is one of the most intensively studied cancer types, partly because of its high prevalence but also because of the existence of its precursor lesions, tubular or villous adenomas, and more recently (sessile) serrated adenomas, which can be detected endoscopically and removed. The morphological steps in the adenoma-carcinoma sequence have been elucidated at a molecular level, which has been facilitated by identification of the genes responsible for familial intestinal cancer. However, apart from early detection of familial forms of CRC and its use in genetic counseling, until recently such detailed molecular knowledge has had little impact on clinical management of the disease. This has dramatically changed in the last decade. With drugs specifically targeting the epidermal growth factor receptor (EGFR) having been shown effective in CRC, mechanisms responsible for resistance have been explored. The finding that KRAS mutated cancers do not respond to anti-EGFR treatment has had a profound impact on clinical management and on molecular diagnostics of CRC. Additional genetic tests for mutations in NRAS, BRAF and PIK3CA contribute to determining who to treat, and others will follow. New therapies effective in patients with advanced CRC are under investigation. Remaining burning questions for optimal management are which patients will relapse after resection of the primary tumor and which patients will respond to the standard 5FU-oxaliplatin adjuvant treatment regimen. Predictive tests to address these issues are eagerly awaited. New classifications of CRC, based on molecular parameters, are emerging, and we will be confronted with new subtypes of CRC, for which the definition is based on combinations of gene expression patterns, chromosomal alterations, gene mutations and epigenetic characteristics. This will be instrumental in designing new approaches for therapy but will also be translated into molecular diagnostics. Both will contribute to improved clinical management of CRC.

Adsorption Behavior of 5-Fluorouracil on Au(111): An In Situ STM Study

The biological effects of chemical substitution of DNA bases triggered several investigations of their physicochemical properties This paper studies the adsorption behavior of a halogenated uracil, 5-fluorouracil (5FU). at the electrochemical interface of Au(111) and sulfuric acid solution. Upon modulation of the electric field across the interface, four distinct phases could be inferred by means of cyclic voltammetry (CV) At negative potentials relative to the SCE electrode, limited by the threshold of hydrogen evolution, no molecular species could be detected by scanning tunneling microscopy (STM) at the reconstructed Au(111)-(23 x root 3) surface, indicating that any physisorbed molecules are randomly distributed Incursion into more positive potentials increases the surface population but doer not form any two-dimensional (2D) physisorbed ordered structure Instead, we observed metastable structures that are only detectable. on surfaces with high defect density At sufficiently high positive potentials. limited by gold oxidation, the molecules are chemisorbed in a (3 x 2 root 3) ordered structure. with the aromatic ring perpendicular to the surface We report the densest chemisorbed monolayer for pyrimidine-derivative molecules (area per molecule 0 14 +/- 0 04 nm(2)). A comparison of the adsorption behavior of uracil derivatives has been made based on recent results of chemical substitution and solvent effects. We propose that pi-stacking is enhanced when halogens are incorporated in the uracil structure, in a similar fashion to what is observed in then crystal structure

Infiltração de 5-fluorouracil no pré-operatório do pterígio

OBJETIVO: Avaliar o efeito do 5-fluorouracil (5FU) injetado intralesionalmente na cabeça do pterígio no período pré-operatório. MÉTODOS: Foram estudados 53 olhos (52 pacientes), sendo 28 pterígios primários e 25 recidivados, divididos em dois grupos: grupo 1 (G1), composto por indivíduos que receberam a injeção de 5-fluorouracil 30 dias antes do procedimento cirúrgico e grupo 2 (G2), no qual o 5-fluorouracil foi injetado 10 dias antes da cirurgia. Todas as cirurgias foram realizadas seguindo-se a mesma técnica cirúrgica, pelo mesmo cirurgião. Os pacientes foram reavaliados 7, 30 e 60 dias após a cirurgia. Os resultados observados foram submetidos à análise estatística. RESULTADOS: A amostra estudada foi constituída por 52,8% de pterígios primários e 47,2% de recidivados, sendo composta igualmente por indivíduos de ambos os sexos. Não ocorreram complicações decorrentes da infiltração da droga. A recidiva foi mais freqüente nos pterígios recidivados e no G1. CONCLUSÃO: O uso intralesional de 5-fluorouracil no pré-operatório do pterígio não provocou efeitos deletérios aos olhos estudados. Houve menor recorrência quando usado o 5-fluorouracil 10 dias antes da exérese cirúrgica, em relação à aplicação 30 dias antes do procedimento.

Efeitos da membrana amniótica homógena associada ao laser arseneto de gálio-alumínio (λ=660) na reparação de mucosite oral em ratos tratados com 5-fluoruracila: análise histológica e imunohistoquímica

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