Evolved bacterial resistance against fluoropyrimidines can lower chemotherapy impact in the Caenorhabditis elegans host.

Metabolism of host-targeted drugs by the microbiome can substantially impact host treatment success. However, since many host-targeted drugs inadvertently hamper microbiome growth, repeated drug administration can lead to microbiome evolutionary adaptation. We tested if evolved bacterial resistance against host-targeted drugs alters their drug metabolism and impacts host treatment success. We used a model system of Caenorhabditis elegans, its bacterial diet, and two fluoropyrimidine chemotherapies. Genetic screens revealed that most of loss-of-function resistance mutations in Escherichia coli also reduced drug toxicity in the host. We found that resistance rapidly emerged in E. coli under natural selection and converged to a handful of resistance mechanisms. Surprisingly, we discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host - only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to host-targeted drugs and by doing so may also impact the host.

Dietary serine-microbiota interaction enhances chemotherapeutic toxicity without altering drug conversion.

The gut microbiota metabolizes drugs and alters their efficacy and toxicity. Diet alters drugs, the metabolism of the microbiota, and the host. However, whether diet-triggered metabolic changes in the microbiota can alter drug responses in the host has been largely unexplored. Here we show that dietary thymidine and serine enhance 5-fluoro 2'deoxyuridine (FUdR) toxicity in C. elegans through different microbial mechanisms. Thymidine promotes microbial conversion of the prodrug FUdR into toxic 5-fluorouridine-5'-monophosphate (FUMP), leading to enhanced host death associated with mitochondrial RNA and DNA depletion, and lethal activation of autophagy. By contrast, serine does not alter FUdR metabolism. Instead, serine alters E. coli's 1C-metabolism, reduces the provision of nucleotides to the host, and exacerbates DNA toxicity and host death without mitochondrial RNA or DNA depletion; moreover, autophagy promotes survival in this condition. This work implies that diet-microbe interactions can alter the host response to drugs without altering the drug or the host.

Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System.

Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU), is sufficient to substantially deplete dividing non-neuronal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99% of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs). More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate application. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cytokine receptors were altered. Individual cytokines or conditioned medium from ß-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtually inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of -brain neuroinflammation.

Postnatal investigation of prenatally induced effects on the vertebral column of rats reduces the uncertainty of classification of anomalies.

Classification of substances as teratogenic is based on the observation of external, visceral and skeletal anomalies. Characterization of anomalies as variation or malformation is contingent upon their postnatal persistence and adversity to health. Lack of information thereof may result in inconsistent or incorrect classification. The aim of this work is the examination of vertebral skeletal anomalies regarding their postnatal fate on PNDs 7 and 21. The anomalies unossified, asymmetric ossification, bipartite ossification, hemicentric, as well as misshapen, did not persist up to PND21 and should be classified as a variation. The finding, cervical vertebra centrum dumbbell-shaped, should be categorized as a malformation due to its continued presence on PND 21. Lumbar centrum supernumerary sinister/dexter/sinister+dexter should also be classified as a malformation. This study demonstrates that postnatal examination is useful and substantially improves the ability to perform a scientifically sound classification of an anomaly compared to investigations terminated on GD 21.

Physicochemical Properties of a Methacrylate-Based Dental Adhesive Incorporated with Epigallocatechin-3-gallate

This study aimed to evaluate the influence of epigallocatechin-3-gallate (EGCG) incorporation on the physicochemical properties of a methacrylate-based dental adhesive. EGCG was added to Adper Easy One (3M-ESPE) except in control group, to obtain concentrations of 0.01% w/w and 0.1% w/w of EGCG-doped adhesives. For water sorption (WS) and solubility (SL) surveys, resin discs were assayed following ISO recommendations (n=10). The degree of conversion (DC) was analyzed by FTIR whereas flexural strength (FS) was tested by three-point bending with bar specimens (n=10). Data were subjected to one-way ANOVA and Tukey's test (p<0.05). No significant difference in the DC, WS and FS were found between the different concentrations of EGCG (p>0.05). Adhesives containing 0.1% or 0.01% of EGCC demonstrated similar values of SL (p>0.05) and higher than those found for adhesive without EGCC (p<0.05). In conclusion, the addition of EGCC to adhesive reduced the solubility without affecting the other evaluated properties.

Esse estudo teve como objetivo avaliar a influência da incorporação de epigalocatequina-3-galato (EGCG) nas propriedades físico-químicas de um sistema adesivo à base de metacrilato. O EGCG foi adicionado ao Adper Easy One (3M-ESPE), exceto para o grupo controle, para a obtenção das concentrações de 0,01% e 0,1% p/p. No ensaio de sorção (S) e solubilidade (SL), foram confeccionados discos de resina de acordo com as recomendações da ISO (n=10). O grau de conversão (GC) foi analisado através de FTIR, enquanto a resistência flexural (RF) foi avaliada em teste de flexão de três pontos com espécimes em forma de barra (n=10). Os dados foram submetidos à Análise de Variância a um critério e teste de Tukey (p<0,05). Não houve diferença significativa entre as concentrações de EGCG testadas no GC, SL e RF (p>0,05). Adesivos contendo EGCG a 0,1% ou 0,01% apresentaram valores similares de SL (p>0,05) e maiores do que os valores obtidos pelo adesivo não incorporado por EGCG. Conclui-se que a adição de EGCG ao adesivo reduziu a solubilidade sem afetar as outras propriedades avaliadas.

Dentin Bond Strength of a Fluoride-Releasing Adhesive System Submitted to pH-Cycling

To evaluate the microtensile bond strength (µTBS) of a fluoride-containing adhesive system submitted to a pH-cycling and storage time regimen for primary outcomes. As secondary outcomes the fluoride released amount was evaluated. Twelve dentin surfaces from sound third molar were divided into 2 groups according to adhesive systems: Clearfil SE Protect (PB) and Clearfil SE Bond (SE). Sticks obtained (1.0 mm2) from teeth were randomly divided into 3 subgroups according to storage regimen model: immediate (24h); 5-month deionized water (W); and pH-cycling model (C). All sticks were tested for µTBS in a universal testing machine. Fluoride concentration was obtained from 1-4 days and 30-day in W and 1-4 days in demineralization (DE)/remineralization (RE) solutions from C, using a fluoride-specific electrode. µTBS and fluoride released data were, respectively, submitted to ANOVA in a split plot design and Tukey, and Friedman' tests (a=0.05). There was no significant interaction between adhesive system and storage regimen for µTBS. W showed the lowest µTBS values. There was no significant difference between 24 h and C models for µTBS. There was no significant difference between adhesive systems. Failure mode was predominantly cohesive within composite for the 24 h and W, for the C group it was mixed for SE and cohesive within composite for PB adhesive system. Fluoride concentrations in the DE/RE solutions were less than 0.03125 ppm and not detected in W. In conclusion, the fluoride-containing adhesive system performed similarly to the regular one. Hydrolytic degradation is the main problem with both adhesive systems, regardless of fluoride contents.

O objetivo principal desse estudo foi avaliar a resistência de união à microtração de dois sistemas adesivos (com e sem flúor) após a ciclagem de pH e armazenagem em água deionizada. A quantidade de flúor liberada foi avaliada secundariamente. Doze terceiros molares hígidos foram separados em 2 grupos de acordo com o sistema adesivo: Clearfil SE Protect - com flúor (PB) e Clearfil SE Bond - sem flúor (SE). Os palitos (1 mm2) obtidos do mesmo dente foram aleatoriamente divididos em 3 subgrupos de acordo com o meio de armazenagem: em água deionizada por 24h ou 5 meses e ciclagem de pH. Os palitos foram tracionados em uma máquina de ensaio universal a 0,5 mm/min. A concentração de flúor foi analisada em água deionizada (1-4 dias e 30 dias) e na solução remineralizadora e desmineralizadora (1-4 dias) usando um eletrodo específico. Os dados de resistência de união e liberação de flúor foram, respectivamente, submetidos à Análise de Variância em esquema de parcela subdividida e ao teste de Friedman (a=0,05). Não houve nenhuma interação significativa na resistência de união entre os sistemas adesivos e os meios de armazenagem. Os menores valores de resistência de união à microtração foram encontrados para os palitos armazenados em água deionizada. Não houve nenhuma diferença significativa nos valores de resistência de união após 24h e ciclagem de pH. Nenhuma diferença significativa na resistência de união foi observada entre os 2 sistemas adesivos. O modo de falha foi predominantemente coesivo em compósito para os grupos armazenados em água por 24h ou 5 meses para ambos os sistemas adesivos. No grupo submetido à ciclagem, a falha foi mista para o SE e coesiva em compósito para o PB. A concentração de flúor nas soluções DE/RE foi menor que 0,03125 ppm e não detectada em água deionizada. Concluindo, o sistema adesivo com flúor (PB) apresentou performance similar ao sistema adesivo sem flúor (SE). A degradação hidrolítica foi o principal fator para ambos os sistemas adesivos, independente da adição de flúor.

Technique of Framework Cemented on Prepared Abutments to Obtain Passive Fit at Fixed Complete Denture: A 2-Year Follow-Up Report

The passive fit of implant-supported dentures is fundamental to the rehabilitation success due the absence of the periodontal ligament in osseointegrated implants. Many techniques to obtain passive fit have been reported in the literature, some inaccessible for the clinicians and dental laboratories. This case report presents a technique to fabricate fixed complete dentures aiming at obtain passive fit with reduced time and cost, but without demerit for the aesthetics, function and longevity. A 40-year-old woman was referred for treatment presenting some teeth in the maxilla and an edentulous mandible, reporting eating problems related to instability and little retention of the mandibular complete denture. Treatment based on the reverse planning was performed to guide the rehabilitation with a complete mandibular fixed complete denture and maxillary occlusal plane adjustment. The framework of the fixed complete denture was manufactured luting a cast metal bar above the prepared titanium cylinder abutments using resin cement. The aim of this technique was to obtain a fixed complete denture with passive fit presenting positive esthetic and functional outcomes after 2 years of follow-up.

A adaptação passiva de próteses implantossuportadas é fundamental para o sucesso da reabilitação devido à inexistência de ligamento periodontal em implantes osseointegrados. Inúmeras técnicas de confecção da infraestrutura destas próteses tem sido relatadas na literatura, algumas inacessíveis para os clínicos e laboratórios de prótese. Este relato de caso apresenta uma técnica para confecção de próteses totais fixas visando obtenção de adaptação passiva com tempo e custo reduzido, porém sem demérito à estética, função e longevidade. Uma paciente de 40 anos se apresentou para tratamento apresentando alguns dentes na maxila e mandíbula edêntula, relatando dificuldades na mastigação relacionados a instabilidade e falta de retenção da prótese total inferior. Foi realizado um planejamento reverso para orientar a reabilitação com prótese total mandibular fixa e adequação do plano oclusal da maxila. A infraestrutura da prótese total fixa foi confeccionada pela cimentação de uma barra metálica em cilindros de titânio preparados com cimento resinoso. O objetivo desta técnica foi obter uma prótese total fixa com adaptação passiva apresentando resultados positivos em termos de estética e função após 2 anos de acompanhamento.

Adenosina trifosfato bioluminescência para avaliação da limpeza de superfícies: uma revisão integrativa / Adenosine triphosphate bioluminescence to evaluate the effectiveness of surface cleaning: an integrative review / Bioluminiscencia adenosina trifosfato para evaluar la eficacia de la limpieza de superficies: una revisión integrativa

Objetivo: Identificar na literatura indicações e controvérsias do ATP bioluminescência para avaliação da efetividade da limpeza de superfícies em estabelecimentos de saúde. Método: Revisão integrativa da literatura, entre 2000 e 2012, nas bases de dados MEDLINE, LILACS, Science Direct, SCOPUS e Isi Web of Knowledge. Resultados: Selecionou-se para esta revisão 15 artigos. O ATP bioluminescência foi apontado como importante recurso educacional e método complementar à inspeção visual e às análises microbiológicas na avaliação da efetividade da limpeza. A impossibilidade de indicar a contaminação da superfície por micro-organismos viáveis, a interferência por substâncias químicas e a dificuldade de interpretação dos resultados constituem as principais controvérsias para o uso deste nos serviços de saúde. Conclusão: Apesar de constituir importante recurso na avaliação da limpeza de superfícies, mais estudos são necessários para incorporação efetiva do método nos serviços de saúde. .

Objective: To identify indications and controversies in the literature of the use of ATP bioluminescence to evaluate the effectiveness of surface cleaning in healthcare facilities. Method: Integrative literature review between 2000 and 2012 in the following databases: MEDLINE, LILACS, Science Direct, SCOPUS and Isi Web of Knowledge. Results: were selected for this review 15 articles. The ATP bioluminescence was considered an important educational resource and complementary method to visual inspection and microbiological evaluation of the effectiveness of cleaning. The impossibility to indicate surface contamination by microorganisms, interference by chemicals and the difficulty of interpreting the results constitute the main controversies in the use of ATP in health services. Conclusion: Although this is an important resource in the evaluation of surface cleaning, more studies are necessary for effective incorporation of the method in health services. .

Objetivo: Identificar en la literatura las indicaciones y controversias sobre el uso de la bioluminiscencia ATP para evaluar la eficacia de la limpieza de superficies en los servicios de salud. Método: Revisión integrativa de la literatura, entre 2000 y 2012, en las siguientes bases de datos: MEDLINE, LILACS, Science Direct, SCOPUS e ISI Web of Knowledge. Resultados: Se seleccionaron para esta revisión 15 artículos. La bioluminiscencia del ATP se considera un importante recurso educativo y método complementario a la inspección visual y la análisis microbiológica de la evaluación de la efectividad de la limpieza. La imposibilidad de indicar contaminación de la superficie por los microorganismos, la interferencia por los productos químicos y la dificultad de interpretar los resultados constituyen las principales controversias en la utilización de ATP en los servicios de salud. Conclusión: Aunque esto es un elemento importante en la evaluación de limpieza de superficies, se necesitan más estudios para incorporación eficaz del método en los servicios de salud. .

Consejería en la detección de VPH como prueba de tamizaje de cáncer cervical: un estudio cualitativo sobre necesidades de mujeres de Michoacán, México / Counseling for HPV detection when used to screen for cervical cancer: a qualitative study on the needs of women from Michoacan, Mexico

Objetivo. Explorar las necesidades de información y consejería de un grupo de mujeres mexicanas al utilizar la prueba de virus de papiloma humano (VPH). Material y métodos. En 2011, se realizaron 24 entrevistas semiestructuradas a mujeres que recibieron el resultado de una prueba de VPH, en dos municipios del estado de Michoacán. El análisis cualitativo de las entrevistas se realizó con las técnicas de la comparación constante. Resultados. Durante el tamizaje, las mujeres recibieron escasa consejería; experimentaron angustia y confusión. Las usuarias de la prueba se mostraron interesadas en recibir información sobre el VPH y el cáncer cervical, el significado de sus resultados, los pasos que habrían de realizar en la atención, apoyo emocional e información vinculada con la transmisión sexual de VPH. Conclusiones. Se requiere diseñar e implementar políticas para impartir educación para la salud y consejería, a la par de la realización de pruebas de VPH.

Objective. To explore the information and counseling needs of a group of Mexican women during use of the HPV test. Materials and methods. In 2011, 24 semistructured interviews were done with women upon receiving HPV test results in two municipalities in the state of Michoacan. Qualitative analysis of the interviews was done using constant comparison techniques. Results. During their use of screening services women received limited counseling; they felt anguish and confusion. Women were interested in receiving information and advice on HPV and cervical cancer, the meaning of test result, next steps to be taken in their healthcare use as well as information and emotional support related to the sexual transmission of HPV. Conclusions. The design and implementation of policies are needed which instigate health education and counseling in conjunction with HPV testing.

Role of activating transcription factor 3 protein ATF3 in necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine.

Necrosis and apoptosis are the two major forms of cell death. We have studied the mechanisms that regulate the cell death observed during treatment of mouse cancer cell line FM3A with the anticancer drug 5-fluoro-2'-deoxyuridine (FUdR). To detect causal differences between necrosis and apoptosis, we exploited the necrosis in original clone F28-7 and the apoptosis in its variant F28-7-A that occur on treatment with FUdR. Activating transcription factor 3 (ATF3) was strongly induced during necrosis but not apoptosis. In addition, we found that ATF3 expression is regulated by heat shock protein 90 (HSP90) at the mRNA stage. Knockdown of Atf3 by siRNA in the F28-7 cells resulted in apoptotic morphology rather than necrotic morphology. These results suggest that ATF3 is a cell-death regulator in necrosis and apoptosis.

UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation.

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.

Combinatorial pharmacologic effects of gemcitabine and its metabolite dFdU.

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.

Association of nuclear-intermediate filament lamin B1 with necrotic- and apoptotic-morphologies in cell death induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analyses. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Our present finding provides an interesting possibility that lamin-B1 may have an important role in regulating cell-death morphology.

Dose-response relationships of rat fetal skeleton variations: Relevance for risk assessment.

In developmental toxicity studies, skeleton abnormalities found in fetuses at term are classified as variations or malformations. The relevance of skeleton variations for human risk assessment, however, is a controversial issue. This paper is a contribution to the discussion on the interpretation of fetal skeleton variations in the context of risk assessment. Dose-response relationships of skeleton variations and malformations induced by three antineoplastic drugs (FUDR: 5-fluoro-2'-deoxyuridine, HU: hydroxyurea and 6-MPr: 6-mercaptopurine-riboside) were evaluated. FUDR (0, 3, 14, 25, 35, 45, 55 and 65mg/kg body wt sc) and HU (0, 250, 300, 350, 400, 450, 500 and 550mg/kg body wt ip) were administered to rats on gestation day 11 (GD 11) while 6-MPr (0, 3, 7, 10 and 14mg/kg body wt sc) was given on GD 11, or on GD 12. Caesarean sections were performed on GD 21 and all fetuses were cleared and stained with alizarin red S for skeleton examination. Drugs given on GD 11 increased the incidence of thoracic and lumbar vertebra (dumbbell-shaped and bipartite ossification center (o.c.) and sternum (misaligned sternebrae) variations in a dose-dependent manner. Occurrence of zygomatic bone fused with maxilla (a variation in our rats) was also increased by HU and 6-MPr (GD 11) but it was not altered by FUDR. Spontaneous occurrence of wavy ribs was reduced by all treatments. Malformations such as cleft palate, tympanic bone absent and tibia absent were also increased in a dose-dependent manner by the three compounds. No observed effect levels (NOEL) for variations, irrespective of the compound administered, were generally lower than NOELs for malformations. In the discussion, we supported the view that any dose-related increase in the incidence of variations should be taken into account for determination of NOELs in routine studies. Increased occurrences of skeleton variations in term fetuses are also to be considered in risk assessment, unless experimental evidence exists that a particular change has no detrimental effect on the animal survival or health after birth or that it does not occur in humans.

The role of floxuridine in metastatic liver disease.

Liver metastases are mainly supplied by the hepatic artery. Sustained high levels of intratumoral drug are achievable with certain drugs given via the hepatic artery. Floxuridine (FUDR) is an ideal drug for hepatic arterial infusion (HAI) due to its short half life, steep dose response curve, high total body clearance, and high hepatic extraction. HAI FUDR has consistently shown higher response rates than systemic chemotherapy alone, and some studies have shown a survival advantage. HAI FUDR in combination with systemic chemotherapy has evolved over the years and may be used in palliative, neoadjuvant, and adjuvant settings. The dramatic responses observed with HAI FUDR plus modern era systemic chemotherapy offer the possibility of resection and cure in selected patients. The high hepatic extraction of FUDR limits systemic side effects. Toxicity includes biliary and gastrointestinal ulcers.

Molecular mechanisms in two cell death-types, necrosis and apoptosis, induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.

Proteome and transcriptome analysis of 5-fluoro-2'-deoxyuridine-induced cell death mechanisms.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. The inhibition of thymidylate synthase causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. We have previously been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. In this report, in order to understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome from F28-7 strain and F28-7-A strain, it was found that ten proteins were increased and six proteins were decreased in F28-7-A strain. Furthermore, transcriptome analysis shows that 127 genes were increased and 181 genes were decreased in F28-7-A strain. These differentially expressed proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. These two techniques clarified numerous features in F28-7 strain and F28-7-A strain. Our results revealed that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.

Relationship between AUC of 5'-DFUR and toxicity of capecitabine, fluoropyrimidine carbamate analogs, and 5'-DFUR in monkeys, mice, and rats.

Capecitabine is an oral fluoropyrimidine carbamate which is converted to 5-fluorouracil (5-FU) via 3 enzymatic step to 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU. We performed 4-week toxicity studies of capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorouridine), galocitabine (trimethoxybenzyl-5'-deoxy-5-fluorocytidine), 4 different fluoropyrimidine carbamate analogs (R=butyl, isopentyl, propyl, or phenethyl), and 5'-DFUR in cynomolgus monkeys with toxicokinetic measurements of intact molecules, 5'-DFCR, and 5'-DFUR. Four-week toxicity data for capecitabine in rats and mice were also obtained for comparison. Capecitabine, galocitabine, butyl, and isopentyl analogs showed similar toxicities in hematopoietic and intestinal organs at 1.0 mmol/kg and the AUCs of 5'-DFUR were approximately 40 to 60 microg*hr/ml. These compounds showed slight toxicity at 0.5 mmol/kg and no toxicity at 0.1 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 5 microg*hr/ml, respectively. Propyl and phenethyl analogs showed slight toxicity at 1.0 mmol/kg and no toxicity at 0.5 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 10 microg*hr/ml, respectively. On the other hand, severe and slight-to-moderate toxicity was observed at 0.5 and 0.25 mmol/kg in 5'-DFUR-treated monkeys and AUCs of 5'DFUR were 35.6 and 5.2 microg*hr/ml, respectively. In mice and rats, the toxicity of capecitabine was less than in monkeys relative to dose, but 5'-DFUR AUCs were almost the same. In conclusion, 5'-DFUR AUC correlated with toxicity following oral administration of capecitabine and its analogs in monkeys, mice, and rats, although this relationship is not seen in humans. Capecitabine was less toxic in monkeys than oral 5'-DFUR according to dose (mmol/kg) and 5'-DFUR AUC.

The relationship of premature mitosis to cytotoxicity in response to checkpoint abrogation and antimetabolite treatment.

Inhibition of one or both of the checkpoint kinases, Chk1 and Chk2, has been proposed as a strategy for improving the efficacy of cytotoxic chemotherapeutic agents in tumor cells. Previous studies have demonstrated that Chk1 inhibition potentiates the cytotoxicity of chemotherapeutic agents in a variety of systems. We designed a study to test whether the simultaneous depletion of Chk1 and Chk2 would sensitize cells to FdUrd- and gemcitabine-induced cytotoxicity to a greater extent than Chk1 depletion alone and to determine the contribution of premature mitosis to cytotoxicity. We found that RNAi-mediated Chk1 depletion enhanced FdUrd- and gemcitabine-mediated cytotoxicity (2- to 3-fold) in Panc-1 and SW620 cells. Furthermore, enhanced cytotoxicity by Chk1 depletion was accompanied by inhibition of FdUrd- or gemcitabine-induced Cdc25A degradation and induction of premature mitotic entry in drug-treated cells. The simultaneous depletion of Chk1 and Chk2 inhibited Cdc25A degradation, induced premature mitotic entry and enhanced cytotoxicity in response to FdUrd and gemcitabine to a similar extent as Chk1 depletion alone. These results imply that Chk2 inhibition has no immediate consequence on survival or cell cycle progression in tumor cells treated with antimetabolites, regardless of their Chk1 status. In addition, these results suggest that premature mitotic entry is a qualitative marker for enhanced antimetabolite-induced cytotoxicity by Chk1 inhibition. The finding that Chk1 inhibition significantly enhanced antimetabolite-induced cytotoxicity supports further investigation and the development of more specific Chk1 inhibitors for use in the clinic.