Minor Histocompatibility Antigen-specific MHC-restricted CD8 T Cell Responses Elicited by Heat Shock Proteins

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2002  |  Pubmed ID: 11823499

In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.

[New Concepts for the Study of Anticancer Drug Resistance]

Bulletin Du Cancer. Jan, 2002  |  Pubmed ID: 11847021

In the past decade, numerous mechanisms of resistance have been described, involving the availability of the drug at the target or the availability of the target itself, but resistance to cell death induction remains far from being understood. The involvement of p53, of Bcl2 and related proteins, of the Fas/Fas-L system and other membrane death receptor pathways, have especially been studied. However, conflicting results have been published concerning the impairment of apoptosis in resistance to cytotoxic drugs. This has shed important doubts on the currently accepted view, which presents apoptosis as a universal determinant of drug activity. These discrepancies are likely to be related to the cell-type specificity of apoptotic pathways and further research is warranted to get a complete picture of the role of cell death inhibition as a drug resistance mechanism. New genetic tools have been recently made available for the study of anticancer drug resistance. Differential or subtractive analyses of gene expression in drug-sensitive and drug-resistant cell lines or tumors have allowed the identification of genes which are potentially responsible for drug resistance, and which had not been recognized previously by the usual analytic approaches. The generation of genetic suppressor elements represents a more functional approach since they can be selected upon the actual resistance properties of the cell lines. Global transcriptome analysis can be performed through the use of cDNA microarrays, either for the comparison of drug-sensitive and resistant cell lines, or for the study of drug effects on gene expression. This may allow the identification of drug-response genes (whose expression is altered by the drug) and of signaling and metabolic pathways involved in drug activity.

Alterations in the Expression of Cytochrome C Oxidase Subunits in Doxorubicin-resistant Leukemia K562 Cells

Biochemical Pharmacology. Mar, 2002  |  Pubmed ID: 11911833

Doxorubicin (DOX), a widely used antitumoral drug, induces numerous modifications in sensitive cells, interacting with nuclear and mitochondrial DNA. In previous studies achieved in two K562 DOX-resistant sublines (K562/0.2R and K562/0.5R), we have shown stable mitochondrial damage comparatively with sensitive parental cells, such as decrease of cytochrome c oxidase activity (COX; EC 1.9.3.1) and cytochrome aa3 content. In order to explain these data, we have studied several COX genes and their expression, in relationship with altered COX activity and multidrug resistance (MDR) phenotype. We have observed a lower expression of the catalytic subunits COX I and II in MDR sublines, which was neither related to mutations in the corresponding mitochondrial genes, nor to a reduced transcription rate. In contrast, we have noticed an increase in both MDR K562 variants, in the mRNA expression of the catalytic subunit COX III, related to an increase in the half-life of these transcripts. Moreover, the doxorubicin resistance phenotype in K562 cells was accompanied by modifications of the expression and steady-state mRNA levels of several nuclear-encoded regulatory COX subunits. Thus, doxorubicin-resistant K562 cells represent an interesting model to study stable modifications concomitant to MDR phenotype. Our results seem to indicate compensatory mechanisms which highlight the complexity of regulatory systems of COX enzyme, involving coordinate regulation of both nuclear and mitochondrial subunit expression.

Effects of Paclitaxel, Cyclophosphamide, Ifosfamide, Tamoxifen and Cyclosporine on the Metabolism of Methoxymorpholinodoxorubicin in Human Liver Microsomes

Cancer Chemotherapy and Pharmacology. Apr, 2002  |  Pubmed ID: 11914905

The effects of paclitaxel, cyclosporine, cyclophosphamide, ifosfamide and tamoxifen on the metabolism of methoxymorpholinodoxorubicin (MMDx), a novel anticancer agent, were investigated using human liver microsomes. Paclitaxel, tamoxifen and cyclosporine dramatically inhibited MMDx metabolism, whereas ifosfamide had only a slight effect at high concentrations (200-300 microM) and cyclophosphamide had no effect. The inhibition was dependent on the concentrations of both MMDx and the coincubated drug. Thus, with 1 microM MMDx, paclitaxel (5 microM), tamoxifen (1 microM) and cyclosporine (1 microM) decreased the metabolic rate of MMDx by 36%, 53% and 62%, respectively. At higher concentrations (10, 5 and 5 microM, respectively, with paclitaxel, tamoxifen and cyclosporine) the inhibition was 52%, 91% and 91%, respectively. These three drugs preferentially inhibited the formation of three metabolites (M2, M3 and M6) among eight metabolites produced in liver microsomes. The inhibitory concentrations of paclitaxel, tamoxifen and cyclosporine on MMDx metabolism were in the range of those observed in patients upon administration of these drugs, which are known to be CYP3A4 substrates. These findings suggest that CYP3A4 drug substrates and MMDx in combination must be used with caution, particularly in view of the fact that MMDx is considered as a prodrug whose activation is entirely dependent upon metabolic transformation by CYP3A4.

Determinants of the Cytotoxicity of Irinotecan in Two Human Colorectal Tumor Cell Lines

Cancer Chemotherapy and Pharmacology. Apr, 2002  |  Pubmed ID: 11914913

Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target.

Determination of Drug Interactions Occurring with the Metabolic Pathways of Irinotecan

Drug Metabolism and Disposition: the Biological Fate of Chemicals. Jun, 2002  |  Pubmed ID: 12019202

Irinotecan or CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] is a derivative of camptothecine used in the treatment of advanced colorectal cancer. It requires activation to SN-38 (7-ethyl-10-hydroxycamptothecine) by carboxylesterase. Irinotecan and SN-38 are detoxified through two major metabolic pathways: the first one leads to oxidative degradation compounds, APC [7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine] and NPC [7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine], and involves cytochrome P450 (3A4 isoform); the second one leads to SN-38 glucuronide (SN-38G) and involves UDP-glucuronosyltransferase (UGT). Using human hepatic microsomes, we studied the interactions of 15 drugs of common use in colorectal cancer patients on these metabolic pathways. Only nifedipine had a significant effect on SN-38 formation, decreasing carboxylesterase activity by 50% at 100 microM and 35% at 10 microM. Three drugs had a significant effect on SN-38G formation: clonazepam increased UGT activity by 50% at 100 microM and 30% at 10 microM, and nifedipine and vinorelbine inhibited the activity by 65 and 55% at 100 microM, respectively, with no effect at 10 microM. Five drugs exerted a significant inhibition on SN-38 formation at 100 microM: clonazepam (70%), methylprednisolone (50%), nifedipine (80%), omeprazole (85%), and vinorelbine (100%). Only omeprazole and vinorelbine still exerted a significant inhibition at 10 microM (30 and 90%, respectively), whereas only vinorelbine had a significant effect at 2 and 0.5 microM (70 and 40%, respectively). In conclusion, potential clinical interactions with the metabolism of irinotecan are likely to be important for vinorelbine, which strongly inhibits irinotecan catabolism by CYP3A4 at clinically relevant concentrations, but not for the other drugs, which exert an effect at concentrations not achievable in patients.

Identification and Characterization of Xenopus CD8+ T Cells Expressing an NK Cell-associated Molecule

European Journal of Immunology. Jun, 2002  |  Pubmed ID: 12115640

Growing evidence suggests that some immune responses are mediated not only by conventional and distinct NK cells and CTL, but also by T cell subsets expressing NK receptors and NK cell-associated molecules. Consistent with our previously published finding that the mAb 1F8 identifies non-T/non-B cells in Xenopus that effect NK-like killing in vitro, we now report that in vivo treatment with this mAb impairs rejection of transplanted MHC class I-negative tumor cells. However, we also find that the NK cell-associated molecule recognized by mAb 1F8 is expressed by a minor population of CD8+ T cells, in which fully rearranged TCRbeta mRNA of at least three different V families can be identified, by contrast, 1F8+/CD8- (NK) cells lack such TCRbeta message. Additionally, the expression of the NK cell-associated molecule can be induced in vitro by a transient submitogenic stimulation of naïve CD8+ T cells with PMA and ionomycin. Such induced expression of 1F8 also occurs in alloantigen-activated CTL and is coincident with a down-regulation of MHC-specific cytotoxicity. Taken together, these new data suggest that regulation of CD8+ T cell activity involving NK cell-associated molecules is a general and evolutionarily ancient phenomenon.

Ontogeny of Xenopus NK Cells in the Absence of MHC Class I Antigens

Developmental and Comparative Immunology. Sep, 2003  |  Pubmed ID: 12798367

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at approximately 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3-4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3-4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRbeta V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.

Evolution of Heat Shock Protein and Immunity

Developmental and Comparative Immunology. Jun-Jul, 2003  |  Pubmed ID: 12697304

Heat shock proteins (hsps) are among the most abundant intracellular proteins. Their synthesis is rapidly up-regulated by various 'stressors' including temperature, glucose deprivation, infection and cancer. Certain hsps are able to: (i). associate and chaperone a large variety of cellular peptides; (ii). be efficiently internalized by antigen presenting cells (APC) through receptor-mediated endocytosis; (iii). channel antigenic peptides they chaperone in the APC's MHC class I presentation pathway; (iv). and stimulate inflammatory cytokines, chemokines and co-stimulatory molecules through the NFkappab signaling pathway. Extracellular release of hsps upon necrotic cell death and their modulated access at the surface of some cells, can be considered as a putative 'danger' signal. Based on the ancient origins and structural conservation of hsps, it has been proposed that, the role of hsps in immunity emerged early in evolution and to be widespread in extant organisms. Data from studies with the frog Xenopus support this proposition.

Signaling FcRgamma and TCRzeta Subunit Homologs in the Amphibian Xenopus Laevis

Developmental and Comparative Immunology. Sep, 2003  |  Pubmed ID: 12798368

The genes encoding FcRgamma and TCRzeta homologs were identified using a bioinformatic approach in the amphibian Xenopus laevis. Deduced amino acid sequence of Xenopus TCRzeta is highly similar to the mammalian and avian counterparts, whereas that of FcRgamma differs by the presence of an additional ITAM-like motif. The presence of the negatively charged residue in the transmembrane regions of both subunits suggests their ability to serve as signal transducing modules in complex with activating receptors. The short extracellular regions contain characteristic cysteine residues responsible for dimerization in the mammalian subunits. According to Southern blot analysis, Xenopus laevis may possess two non-allelic genes for each subunit. Northern blots revealed FcRgamma transcripts of two sizes differentially expressed in thymus, spleen, intestine, liver and kidney. TCRzeta mRNA was predominantly expressed in the thymus and spleen. These data indicate that the amphibian immune system employs activating receptor complexes arranged in a mammalian-like way.

Development and Characterization of a Model System to Study Amphibian Immune Responses to Iridoviruses

Virology. Jul, 2003  |  Pubmed ID: 12842616

The recent realization that viruses within the family Iridoviridae may contribute to the worldwide decline in amphibians makes it urgent to understand amphibian antiviral immune defenses. We present evidence that establishes the frog Xenopus laevis as an important model with which to study anti-iridovirus immunity. Adults resist high doses of FV3 infection, showing only transitory signs of pathology. By contrast, naturally MHC class-I-deficient tadpoles are highly susceptible to FV3 infection. Monitoring of viral DNA by PCR indicates a preferential localization of FV3 DNA in the kidney, with the inbred MHC homozygous J strain appearing to be more susceptible. Clearance of virus as measured by detection of FV3 DNA and also the disappearance of pathological and behavioral symptoms of infection, acceleration of viral clearance, and detection of IgY anti-FV3 antibodies after a second injection of FV3 are all consistent with the involvement of both cellular and humoral adaptive antiviral immune responses.

Multidrug Resistance Reversal Agents

Journal of Medicinal Chemistry. Nov, 2003  |  Pubmed ID: 14584929

Bacterial Stimulation Upregulates the Surface Expression of the Stress Protein Gp96 on B Cells in the Frog Xenopus

Cell Stress & Chaperones. 2003  |  Pubmed ID: 14984060

The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherichia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.

Cellular Parameters Predictive of the Clinical Response of Colorectal Cancers to Irinotecan. A Preliminary Study

Anticancer Research. Mar-Apr, 2004  |  Pubmed ID: 15160997

We have examined, in this study, the feasibility of determining cellular factors contributing to irinotecan activity in colorectal cancers. Irinotecan is a camptothecin derivative requiting carboxylesterase activation to SN-38, which interacts with its target enzyme, topoisomerase I.

Anti-tumor MHC Class Ia-unrestricted CD8 T Cell Cytotoxicity Elicited by the Heat Shock Protein Gp96

European Journal of Immunology. Sep, 2004  |  Pubmed ID: 15307177

In Xenopus as in mammals, gp96 stimulates MHC-restricted cellular immunity against chaperoned minor histocompatibility (H) antigens (Ag). In adult Xenopus, gp96 also elicits peptide-specific effectors against MHC class Ia-negative 15/0 tumors. To determine whether gp96 can generate functionally heterogeneous CD8+ effectors (CTL that kill MHC class Ia+ minor H-Ag-disparate lymphoblasts and MHC class Ia- tumor targets), LG-6 isogenetic frogs were immunized with gp96 purified either from MHC-identical but minor H-Ag-disparate LG-15 normal tissues or from the MHC class Ia-negative 15/0 tumor line (derived from LG-15 frogs). LG-15 normal liver-derived gp96 did not induce detectable CD8+ in vitro killing against 15/0 tumor cells. However, 15/0-derived gp96 did induce killing against both MHC class Ia+ LG-15 lymphoblasts and the MHC class Ia- 15/0 tumor, but not against another MHC class Ia- tumor (B3B7) or against LG-6 lymphoblasts. Tumor killing was better when 15/0 rather than normal LG-15 irradiated stimulators were used, but in vitro stimulation without prior in vivo immunization was ineffective. These data suggest that (1) 15/0-derived gp96 chaperones minor H-Ag shared with normal LG-15 lymphocytes and elicits MHC-restricted CTL, and (2) 15/0-derived gp96, but not normal liver-derived gp96, generates CD8+ effectors that kill 15/0 tumor cells in the absence of MHC class Ia expression.

Predicting Drug Response Based on Gene Expression

Critical Reviews in Oncology/hematology. Sep, 2004  |  Pubmed ID: 15331079

Predicting drug response is a challenging problem in oncology. In the 1975-1985 decade, important efforts were devoted to the generation of cellular assays able to predict, on an individual basis, the in vitro response of tumour cells to chemotherapeutic agents, but such methods could not be adopted in routine. Numerous mechanisms of resistance to anticancer agents have been identified in cultured cell lines selected for growth in the presence of infratoxic, increasing doses of anticancer agents. They mainly concern drug transport, drug activation or detoxification, target quantitative or qualitative alterations, DNA repair efficiency, and alterations in signalling and/or execution of cell death programmes. New molecular biology techniques have been developed in order to identify the genes involved in drug resistance; they mainly involve differential expression techniques, but functional approaches may also prove informative. The availability of techniques of gene expression profiling has allowed to establish correlations between gene expression and drug sensitivity of tumour cells or human cancers. This type of approach has been initiated on in vitro systems by the National Cancer Institute (NCI) in the USA and is pursued by a growing number of public and private laboratories around the world. In the clinical setting, a number of genes or proteins have been identified as potential predictive markers of drug activity and their use could be progressively implemented for drug selection in patients receiving chemotherapy, allowing thus more rational and individualised treatments.

Pharmacogenetics of Human Carboxylesterase 2, an Enzyme Involved in the Activation of Irinotecan into SN-38

Clinical Pharmacology and Therapeutics. Dec, 2004  |  Pubmed ID: 15592324

Irinotecan, a drug widely used in the treatment of advanced colorectal cancers, is a prodrug requiring activation to 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterase 2 (hCE2). The existence of functional polymorphisms in the gene encoding this enzyme could explain the individual variability in drug efficacy and toxicity. We have explored this possibility in looking for single nucleotide polymorphisms and their functional consequence.

Phylogenetic Conservation of Gp96-mediated Antigen-specific Cellular Immunity: New Evidence from Adoptive Cell Transfer in Xenopus

Transplantation. Nov, 2004  |  Pubmed ID: 15599304

In vertebrates from man to frogs, the heat shock protein (hsp) gp96 elicits T-cell responses against antigenic peptides that it chaperones. In Xenopus, immunization with gp96 purified from normal tissues accelerates rejection of MHC identical, minor histocompatibility (H) antigen-disparate skin grafts in vivo and induces MHC-restricted CTL responses in vitro. Also in Xenopus, gp96 derived from MHC class I-negative tumors elicits peptide-specific responses against these tumors in vivo and MHC-unrestricted CD8 killing in vitro. We have developed an adoptive cell transfer protocol to further characterize these gp96-stimulated Xenopus effectors in vivo.

MS-209 Schering

Current Opinion in Investigational Drugs (London, England : 2000). Dec, 2004  |  Pubmed ID: 15648956

MS-209, a quinolone-derived sphingomyelin synthase inhibitor that blocks P-glycoprotein and multidrug resistance-associated protein-1, is under development by Schering for the potential treatment of multidrug resistant tumors. By March 2003, phase II trials in breast cancer and non-small-cell lung cancer had been initiated.

Quantification of the Expression of Multidrug Resistance-related Genes in Human Tumour Cell Lines Grown with Free Doxorubicin or Doxorubicin Encapsulated in Polyisohexylcyanoacrylate Nanospheres

Anticancer Research. Nov-Dec, 2004  |  Pubmed ID: 15736412

Doxorubicin (dox) encapsulated in polyisohexylcyanoacrylate nanospheres (PIHCA-dox) can circumvent P-glycoprotein-mediated multidrug resistance (MDR). In order to investigate whether this drug formulation is able to select MDR cells in culture in the same way as free doxorubicin does, two human tumour cell lines, K562 and MCF7, were grown with increasing concentrations of either free dox or PIHCA-dox. For both drug formulations and for each selection level, the cell lines were more resistant to free dox than to PIHCA-dox. The MCF7 sublines selected with PIHCA-dox exhibited a higher level of resistance to both doxorubicin formulations than those selected with free doxorubicin. Different levels of overexpression of several genes involved in drug resistance (MDR1, MRP1, BCRP and TOP2alpha) occurred in the resistant variants. MDR1 gene overexpression was consistently higher in free dox-selected cells than in PIHCA-dox-selected cells, while this was the reverse for the BCRP gene. Overexpression of the MRP1 and TOP2alpha genes was also observed in the selected variants. Our results show that several mechanisms may be involved in the acquisition of drug resistance and that drug encapsulation markedly alters or delays these processes.

Xenopus As an Experimental Model for Studying Evolution of Hsp--immune System Interactions

Methods (San Diego, Calif.). Jan, 2004  |  Pubmed ID: 14624877

The frog Xenopus provides a unique model system for studying the evolutionary conservation of the immunological properties of heat shock proteins (hsps). General methods for maintaining and immunizing isogenetic clones of defined MHC genotypes are presented together with more recently developed protocols for exploring hsp-mediated immune responses in vitro (proliferative and cytotoxic assays) and in vivo (adoptive cell transfer and antibody treatment) in adults and in naturally MHC class I-deficient larvae. Finally, techniques to study modalities of expression of the endoplasmic reticulum resident gp96 at the cell surface of tumor and normal lymphocytes are considered.

Molecular Determinants of the Cytotoxicity of Platinum Compounds: the Contribution of in Silico Research

Cancer Research. Jan, 2004  |  Pubmed ID: 14729645

Gene expression profiling of tumors allows the establishment of relationships between gene expression profiles and sensitivity to anticancer drugs. In an attempt to study the molecular determinants of the activity of platinum compounds, we explored the publicly available databases of the National Cancer Institute (NCI; http://dtp.nci.nih.gov), which allow access to the gene expression profiles of the 60 cell lines for which drug cytotoxicity patterns already existed. Using this database, we have conducted an in silico research to identify the genes the expression of which was positively or negatively correlated to the sensitivity to four platinum compounds (cisplatin, carboplatin, oxaliplatin and tetraplatin). Important similarities were noticed between cisplatin and carboplatin on one hand, and tetraplatin and oxaliplatin on the other hand. In the restricted panel of 1416 genes and molecular markers, we identified 204 markers, among which 120 corresponded to identified genes, that significantly correlated (P < 0.001) with the cytotoxicity of at least one platinum compound. For example, the functionality of the p53-activated pathway appeared positively correlated with the cytotoxicity of all platinum compounds. More specific are the positive correlations between RAS gene mutations and MYC expression and the cellular sensitivity to oxaliplatin. Among the parameters already known as related to the sensitivity to platinum compounds, we identified, in the complete set of 9400 genes, numerous significant relationships, such as the negative correlations between ERB-B2 and BCL-X(L) expressions and the cytotoxicity of the platinum compounds. Public databases mining, therefore, appears to be a valuable tool for the identification of determinants of anticancer drug activity in tumors.

[Recent Advances in Pharmacogenomics in Oncology]

Bulletin Du Cancer. Jan, 2004  |  Pubmed ID: 14975802

Progress in understanding the molecular mechanisms of oncogenesis, together with the sequencing of the human genome and the availability of huge databases, bring new tools for the discovery and the evaluation of new potential targets for cancer therapy and for the individualisation of cancer chemotherapy as a function of the molecular characteristics of tumours. Targeting the genetic alterations of cancer cells appears feasible and the first successes of this approach allow to remain optimistic about the renewal of our therapeutic armamentarium. In addition, seeking for correlations between gene expression profiles and chemosensitivity has been performed on the in vitro models of the National Cancer Institute and may allow crucial improvements in the identification of patients who world best take advantage of a specific chemotherapy. Clinical trials, first on a retrospective basis, then performed prospectively, are implemented to validate this approach.

Predicting Drug Response and Toxicity Based on Gene Polymorphisms

Critical Reviews in Oncology/hematology. Jun, 2005  |  Pubmed ID: 15890268

The sequencing of the human genome has allowed the identification of thousands of gene polymorphisms, most often single nucleotide polymorphims (SNP), which may play an important role in the expression level and activity of the corresponding proteins. When these polymorphisms occur at the level of drug metabolising enzymes or transporters, the disposition of the drug may be altered and, consequently, its efficacy may be compromised or its toxicity enhanced. Polymorphisms can also occur at the level of proteins directly involved in drug action, either when the protein is the target of the drug or when the protein is involved in the repair of drug-induced lesions. There again, these polymorphisms may lead to alterations in drug efficacy and/or toxicity. The identification of functional polymorphisms in patients undergoing chemotherapy may help the clinician prescribe the optimal drug combination or schedule and predict with more accuracy the response to these prescriptions. We have recorded in this review the polymorphisms that have been identified up till now in genes involved in anticancer drug activity. Some of them appear especially important in predicting drug toxicity and should be determined in routine before drug administration; this is the case of the most common variations of thiopurine methyltransferase for 6-mercaptopurine and of dihydropyrimidine dehydrogenase for fluorouracil. Other appear determinant for drug response, such as the common SNPs found in glutathione S-transferase P1 or xereoderma pigmentosum group D enzyme for the activity of oxaliplatin. However, confusion factors may exist between the role of gene polymorphisms in cancer risk or overall prognosis and their role in drug response.

Major Improvement of the Reference Method of the French Drug Resistance Network for P-glycoprotein Detection in Human Haematological Malignancies

Leukemia Research. Sep, 2005  |  Pubmed ID: 16038729

The aim of this study was to improve significantly the sensitivity and specificity of the flow cytometric assay of P-glycoprotein (Pgp) implemented and validated by the laboratories of the French Drug Resistance Network [Huet S, Marie JP, Gualde N, Robert J. Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network. Cytometry 1998;34:248-56] in cells displaying low level of resistance. Fluoresceine-conjugated monoclonal antibodies (Mabs) and propidium iodide were respectively replaced by phycoerythrin-conjugated Mabs and Sytox green. The removal of erythrocytes and granulocytes by density gradient was replaced by the lysis of erythrocytes after Mab incubation. Using these conditions, Pgp could be detected in the K-H30 line, which was negative in former studies, with Mab/Control ratios increasing by 3.7- to 5.9-fold, and Mab/Control ratios in the parental sensitive K562 line still ranging between 0.8 and 1.2. When tested on 16 blood samples from patients presenting haematological malignancies, six samples presented low positivity, which was not detected with the former method, while 10 samples remained negative with the two methods. Pgp was specifically detected in pathological blood cells in the six positive samples.

8-O-Azeloyl-14-benzoylaconine: a New Alkaloid from the Roots of Aconitum Karacolicum Rapcs and Its Antiproliferative Activities

Bioorganic & Medicinal Chemistry. Dec, 2005  |  Pubmed ID: 16081293

A new alkaloid of Aconitum karacolicum Rapcs, from the Ranunculaceae family, collected in Kirghizstan, was isolated from the roots of this plant, using a purification scheme based upon its in vitro antiproliferative properties against three human tumour cell lines in culture. Structural identification was performed using high resolution MS-MS mass spectrometry and (1)H, (13)C, 2D NOESY NMR spectroscopy analysis. This compound consists of a 14-benzoylaconine moiety substituted on C-8 by an azeloyl chain. It presents in vitro cytotoxicity with an IC(50) of about 10-20 microM, which warrants further investigation on its possible interest in cancer chemotherapy.

Adaptive Immunity and Histopathology in Frog Virus 3-infected Xenopus

Virology. Feb, 2005  |  Pubmed ID: 15680432

Xenopus has been used as an experimental model to evaluate the contribution of adaptive cellular immunity in amphibian host susceptibility to the emerging ranavirus FV3. Conventional histology and immunohistochemistry reveal that FV3 has a strong tropism for the proximal tubular epithelium of the kidney and is rarely disseminated elsewhere in Xenopus hosts unless their immune defenses are impaired or developmentally immature as in larvae. In such cases, virus is found widespread in most tissues. Adults, immunocompromised by depletion of CD8+ T cells or by sub-lethal gamma-irradiation, show increased susceptibility to FV3 infection. Larvae and irradiated (but not normal) adults can be cross-infected through water by infected adult conspecifics (irradiated or not). The natural MHC class I deficiency and the absence of effect of anti-CD8 treatment on both larval CD8+ T cells and larval susceptibility to FV3 are consistent with an inefficient CD8+ T cell effector function during this developmental period.

CD91 Up-regulates Upon Immune Stimulation in Xenopus Adult but Not Larval Peritoneal Leukocytes

Immunogenetics. Jan, 2005  |  Pubmed ID: 15592667

CD91, the endocytic receptor for alpha2-macroglobulin (alpha2M), mediates the internalization of certain heat shock proteins (hsps) and the cross-presentation of peptides they chaperone by antigen-presenting cells. The phylogenetic conservation of the immunologically active CD91 ligands, alpha2M and hsps, is consistent with the idea of an ancestral system of immune surveillance. We have further explored this hypothesis by taking advantage of the frog Xenopus, and asked how conserved is CD91 and whether the expression of CD91 is differentially modulated during immune responses of class I-positive adult and naturally class I-negative larvae. We have identified a Xenopus CD91 gene homologue that displays high sequence identity (>65%) with other CD91 homologues and contains an additional distinctive cytoplasmic NPXY motif. Phylogenetic analysis indicates that CD91 homologues branch as a monophyletic group distinct from other LDLRs; this suggests an origin of CD91 contemporary with that of metazoans. A 14-kb transcript is detected by Northern blotting in most adult and larval tissues, including lymphoid tissues. RT-PCR study reveals that CD91 is expressed in most cell types, including adult macrophages, B and T cells as well as in splenocytes and thymocytes from naturally MHC class I negative larvae. CD91 is markedly up-regulated in vivo by adult peritoneal leukocytes following bacterial and viral stimulation; it is constitutively expressed on class I-negative larval peritoneal leukocytes at high levels and cannot be further upregulated by such stimulation. These data are in agreement with a conserved role of CD91 in immunity.

Experimental Study of Dexrazoxane-anthracycline Combinations Using the Model of Isolated Perfused Rat Heart

Toxicology Letters. Feb, 2006  |  Pubmed ID: 16129573

We have studied the protective effect of dexrazoxane on the cardiac toxicity induced by the anthracyclines currently used in clinics, doxorubicin, epirubicin, daunorubicin and idarubicin, with special emphasis on determining the optimal dose of dexrazoxane. This was performed using the model of isolated perfused rat heart after 12-day combination treatment of anthracyclines used at equi-cardiotoxic doses, and dexrazoxane used at 10-fold or 20-fold the anthracycline dose. We have shown in this study that dexrazoxane by itself was not cardiotoxic, and was able to significantly reduce anthracycline cardiac toxicity without increasing the general toxicity induced by these drugs. Using dexrazoxane at 20 times the anthracycline dose provided a better cardioprotection than using it at 10 times the anthracycline dose; at the higher dexrazoxane dose, the functional cardiac parameters (developed pressure, contractility and relaxation) were not different from those recorded in control animals.

Generation of a Long-lasting, Protective, and Neutralizing Antibody Response to the Ranavirus FV3 by the Frog Xenopus

Developmental and Comparative Immunology. 2006  |  Pubmed ID: 16380162

Xenopus serves as an experimental model to evaluate the contribution of adaptive immunity in host susceptibility to emerging ranaviral diseases that may contribute to amphibian population declines. It has been previously shown that following a secondary infection with the ranavirus frog virus 3 (FV3), adult Xenopus more rapidly clear FV3 and generate specific anti-FV3 IgY antibodies. We have further evaluated the potency and persistence of the Xenopus antibody response against FV3. Frogs inoculated with FV3 (without adjuvant) up to 15 months after priming produce specific, thymus-dependent anti-FV3 IgY antibodies detectable from 10 days to 8 weeks post-infection. These antisera from boosted frogs are neutralizing in vitro and provide partial passive protection to susceptible larvae when they are injected a few minutes before FV3 inoculation. These results with Xenopus suggest that other anuran amphibians are likely to develop effective long-lasting protective humoral immunity after an initial viral exposure.

Mitochondrial Localization and Activity of P-glycoprotein in Doxorubicin-resistant K562 Cells

Biochemical Pharmacology. Apr, 2006  |  Pubmed ID: 16499877

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

Genetic Polymorphisms of the XPG and XPD Nucleotide Excision Repair Genes in Sarcoma Patients

International Journal of Cancer. Journal International Du Cancer. Oct, 2006  |  Pubmed ID: 16646069

There are more than 50 subtypes of soft tissue sarcomas, among which 30% are associated with specific genetic alterations, including translocations. Several studies have reported associations between cancer risk and polymorphisms of DNA repair genes from the nucleotide excision repair (NER) pathway. NER involves more than 20 proteins whose inactivation leads to xeroderma pigmentosum (XP) or cockayne syndrome (CS), among which XPD, a helicase allowing DNA strand excision by the endonuclease XPG. DNA from 93 patients with synovial sarcomas, myxoid liposarcomas, dermatofibrosarcomas protuberans (DFSP), malignant fibrous histiocytomas and leiomyosarcomas were genotyped for both XPD Lys751Gln and XPG Asp1104His polymorphisms. Departure from Hardy-Weinberg was highly significant for the XPG polymorphism with an excess of heterozygotes in synovial sarcomas (p = 1.5 x 10(-5)), myxoid liposarcomas (p = 1.5 x 10(-4)) and to a lesser extent in DFSP (p = 0.028). In the case of XPD, a significant deviation was observed in synovial sarcomas (p = 3 x 10(-6)) and DFSP (p = 0.0014). When tumors were pooled according to their genetic alterations, the proportion of carriers of the variant XPG allele was significantly increased in sarcomas with specific translocations as compared to sarcomas with complex genetics (p < 10(-9)). No difference was found for XPD. Genotyping of the tumor samples in synovial sarcomas and myxoid liposarcomas revealed frequent loss of heterozygosity for XPG, mostly due to the loss of the frequent allele. For XPD, both alleles were lost with a similar frequency. Our results raise the potential implication of the XPG Asp1104His polymorphism in the occurrence of chromosomal translocations associated with specific subtypes of sarcomas.

A Computational Model Integrating Electrophysiology, Contraction, and Mitochondrial Bioenergetics in the Ventricular Myocyte

Biophysical Journal. Aug, 2006  |  Pubmed ID: 16679365

An intricate network of reactions is involved in matching energy supply with demand in the heart. This complexity arises because energy production both modulates and is modulated by the electrophysiological and contractile activity of the cardiac myocyte. Here, we present an integrated mathematical model of the cardiac cell that links excitation-contraction coupling with mitochondrial energy generation. The dynamics of the model are described by a system of 50 ordinary differential equations. The formulation explicitly incorporates cytoplasmic ATP-consuming processes associated with force generation and ion transport, as well as the creatine kinase reaction. Changes in the electrical and contractile activity of the myocyte are coupled to mitochondrial energetics through the ATP, Ca2+, and Na+ concentrations in the myoplasmic and mitochondrial matrix compartments. The pseudo steady-state relationship between force and oxygen consumption at various stimulus frequencies and external Ca2+ concentrations is reproduced in both model simulations and direct experiments in cardiac trabeculae under normoxic conditions, recapitulating the linearity between cardiac work and respiration in the heart. Importantly, the model can also reproduce the rapid time-dependent changes in mitochondrial NADH and Ca2+ in response to abrupt changes in workload. The steady-state and dynamic responses of the model were conferred by ADP-dependent stimulation of mitochondrial oxidative phosphorylation and Ca2+ -dependent regulation of Krebs cycle dehydrogenases, illustrating how the model can be used as a tool for investigating mechanisms underlying metabolic control in the heart.

Structural Phylogenetic Analysis of Activation-induced Deaminase Function

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2006  |  Pubmed ID: 16785531

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.

Relationships Between Genetic Polymorphisms and Anticancer Drug Cytotoxicity Vis-à-vis the NCI-60 Panel

Pharmacogenomics. Sep, 2006  |  Pubmed ID: 16981845

The National Cancer Institute (NCI)-60 panel consists of 60 human tumor cell lines initially established for screening thousands of molecules for antiproliferative activity. It has been powerful for deciphering the relationships between anticancer drug cytotoxicity and cell molecular characteristics. We tested its potential interest for establishing relationships between the polymorphism of genes involved in drug metabolism and transport or in DNA repair, and drug cytotoxicity extracted from NCI databases.

Characterization of Primary and Memory CD8 T-cell Responses Against Ranavirus (FV3) in Xenopus Laevis

Journal of Virology. Mar, 2007  |  Pubmed ID: 17182687

In mammals, resistance to primary and secondary viral infections critically involves major histocompatibility complex class I-restricted cytotoxic CD8+ T lymphocytes (CTLs). Although many gene homologues involved in CTL function have been identified in all vertebrate classes, antiviral CTL responses have been poorly characterized for ectothermic vertebrates. Because of the threat of emerging wildlife viral diseases to global biodiversity, fundamental research on comparative viral immunity has become crucial. Ranaviruses (family Iridoviridae) are double-stranded DNA viruses possibly implicated in the worldwide decline of amphibian populations. We used the frog Xenopus laevis as a model to evaluate adaptive immune responses to the ranavirus frog virus 3 (FV3). FV3 infects the kidneys of adults but is cleared within 4 weeks, with faster clearance upon secondary infections. In vivo depletion of CD8+ T cells markedly decreases the survival of adults after viral infection. To further investigate the involvement of anti-FV3 CD8+ T-cell effectors in host resistance in vivo, we determined the proliferation kinetics of CD8+ T cells in the spleen by bromodeoxyuridine incorporation and their infiltration of kidneys by immunohistology. Upon primary infection, CD8+ T cells significantly proliferate in the spleen and accumulate in infected kidneys from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide in vivo evidence of protective antigen-dependent CD8+ T-cell proliferation, recognition, and memory in fighting a natural pathogen in Xenopus.

In Vivo Study of T-cell Responses to Skin Alloantigens in Xenopus Using a Novel Whole-mount Immunohistology Method

Transplantation. Jan, 2007  |  Pubmed ID: 17264812

The African clawed frog, Xenopus, is a widely used comparative model for studying the immune response to transplantation antigens.

Involvement of Gene Polymorphisms of Thymidylate Synthase in Gene Expression, Protein Activity and Anticancer Drug Cytotoxicity Using the NCI-60 Panel

European Journal of Cancer (Oxford, England : 1990). Mar, 2007  |  Pubmed ID: 17317154

A significant association has been established, in clinical studies, between the expression or activity of thymidylate synthase (TYMS) and the efficiency of fluorouracil. TYMS expression is partly under the dependence of gene polymorphisms in the 5' and 3' untranslated regions (UTR), but conflicting results have been obtained about their roles on fluorouracil efficiency. In this study, we wanted to use the National Cancer Institute (NCI) panel of 60 human tumour cell lines to clarify this problem. Three relevant polymorphisms of the TYMS gene were studied: (i) the 5'UTR tandem repeat of 28-bp (2R/3R polymorphism); (ii) the single nucleotide polymorphism (SNP) within the second repeat (3C/3G polymorphism); (iii) the 3'UTR 6-bp deletion (+6/-6 polymorphism). Allele frequencies were close to those expected in a Caucasian population (2R/3C/3G: 53/29/18%; +6/-6: 68/32%), but the proportion of heterozygous genotypes was lower than expected from allele frequencies. The 2R and 3G alleles were significantly associated with the +6 and the -6 alleles, respectively. There was a significant association between the presence of the 3G allele and TYMS mRNA expression and catalytic activity, particularly in p53-mutated cell lines. However, no significant correlation existed between fluorouracil cytotoxicity, as extracted from the NCI databases, and TYMS expression, activity or polymorphisms.

Inhibition of Topoisomerase I Cleavage Activity by Thiol-reactive Compounds: Importance of Vicinal Cysteines 504 and 505

The Journal of Biological Chemistry. May, 2007  |  Pubmed ID: 17355975

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.

Functional Analysis of the Gene Expression Profiles of Colorectal Cancer Cell Lines in Relation to Oxaliplatin and Cisplatin Cytotoxicity

Oncology Reports. May, 2007  |  Pubmed ID: 17390068

The objective was to relate the gene expression profiles of colorectal cancer cells in culture to the in vitro cytotoxicity of cisplatin and oxaliplatin. We studied the gene expression profiles of six human colorectal cancer cell lines, using the Atlas Plastic Human 8K Microarray from Clontech, and related it to the in vitro cytotoxicities of oxaliplatin and cisplatin obtained by inhibition of exponential growth of cells. We calculated the Pearson's coefficients of correlation (r) between gene expression and drug IC50. A functional analysis was performed using the Gene Ontology Consortium database. Results were validated on a series of representative genes by real-time quantitative PCR. Validation of the significance of the coefficients of correlation was also performed using a leave-one-out analysis. We identified 394 genes whose expression was significantly correlated (P<0.05) to oxaliplatin cytotoxicity and 40 with cisplatin cytotoxicity. Three major functions were preferentially involved in oxaliplatin activity: protein synthesis, cell energetics and response to oxidative stress. No significant correlation was observed between oxaliplatin or cisplatin cytotoxicity and the expression of genes involved in DNA repair, cell proliferation or cell adhesion. A strongly significant correlation was found between the microarray and the rt-PCR approaches (r=0.968, P<10(-6)). The leave-one-out analysis showed that the same functions still appeared significantly involved in the activity of both drugs. Based on the functional analysis, we hypothesized that oxaliplatin would specifically form protein adducts during synthesis, thus exposing their thiol groups, which are known to be especially vulnerable to reactive oxygen species.

Involvement of Nonclassical MHC Class Ib Molecules in Heat Shock Protein-mediated Anti-tumor Responses

European Journal of Immunology. Jun, 2007  |  Pubmed ID: 17492621

Nonclassical MHC class Ib (class Ib) genes are found in all jawed vertebrates, and their products are hypothesized to be indicators of intracellular stress and malignancy. They may be involved in immune recognition of classical MHC class Ia (class Ia)-low or -negative tumor cells through their interaction with T cell receptors and/or non-T cell inhibitory or triggering receptors expressed by NK cells and T cells. In the frog Xenopus, the molecular chaperone gp96 mediates a potent immune response involving antigen-specific classical class Ia-unrestricted CD8+ CTL (CCU-CTL) against a transplantable thymic tumor (15/0) that does not express class Ia molecules. We hypothesized that Xenopus nonclassical class Ib gene products (XNC) are involved in gp96-mediated CCU-CTL anti-tumor responses. To investigate the involvement of class Ib gene products in Xenopus anti-tumor responses, we generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing short hairpin RNA (shRNA) to silence either XNC directly or beta2m to prevent class Ib surface expression. Both types of 15/0 transfectants are more resistant to CCU-CTL killing, more tumorigenic and more susceptible to NK-like cell killing. This study provides in vitro and in vivo evidence of the evolutionary conservation of class Ib involvement in anti-tumor CD8+ T cell responses.

Long-term and Short-term Models for Studying Anthracycline Cardiotoxicity and Protectors

Cardiovascular Toxicology. 2007  |  Pubmed ID: 17652818

The clinical importance of the cardiotoxicity of anthracyclines requires the availability of preclinical models able to predict the cardiotoxicity of novel anthracycline analogs in reference to doxorubicin or of cardioprotectors aimed at circumventing the deleterious effects of these drugs. The reference model has been defined long ago and has proven its validity. Weanling rabbits given weekly injections of doxorubicin for 4 months developed a cardiomyopathy, which can be assessed from a clinical and pathological point of view. Models in other animals such as rats or mice were similarly implemented, also with long-term exposures to the drug, resulting in cardiac failure and severe pathological alterations, which could be graded for comparison. Starting from the evidence that the damage caused by anthracyclines on cardiomyocytes was immediate after each injection and that the functional efficiency of the myocardium should be affected long before the morphological alterations become detectable, we developed a short-term model studying the cardiac performances of isolated perfused hearts of rats that had been treated within 12 days by repetitive administrations of the molecule(s) to be tested. This model provided the data expected from clinical experience: epirubicin appeared less cardiotoxic than doxorubicin; liposomal formulations appeared less cardiotoxic than free drug formulations; dexrazoxane strongly protected against doxorubicin cardiotoxicity. We were then able to show that paclitaxel could potentialize doxorubicin cardiotoxicity, but that docetaxel did not so; or that a high dose of dexrazoxane brought significantly higher protection than a conventional dose. Based upon these contributions, we can encourage the use of the short-term model of isolated perfused rat heart to screen the preclinical cardiotoxicity of anthracycline molecules, formulations and combinations.

A Phase II Study for the Evaluation of Quinine As a Modulator of Multidrug Resistance in Non-Hodgkin's Lymphoma

European Journal of Cancer (Oxford, England : 1990). Jan, 2007  |  Pubmed ID: 16824747

Conservation of IL-6 Trans-signaling Mechanisms Controlling L-selectin Adhesion by Fever-range Thermal Stress

European Journal of Immunology. Oct, 2007  |  Pubmed ID: 17823890

Fever is associated with improved survival during infection in endothermic and ectothermic species although the protective mechanisms are largely undefined. Previous studies indicate that fever-range thermal stress increases the binding activity of the L-selectin homing receptor in human or mouse leukocytes, thereby promoting trafficking to lymphoid tissues across high endothelial venules (HEV). Here, we examined the evolutionary conservation of thermal regulation of L-selectin-like adhesion. Leukocytes from animals representing four taxa of vertebrates (mammals, avians, amphibians, teleosts) were shown to mediate L-selectin-like adhesion under shear to MECA-79-reactive ligands on mouse HEV in cross-species in vitro adherence assays. L-selectin-like binding activity was markedly increased by fever-range thermal stress in leukocytes of all species examined. Comparable increases in L-selectin-like adhesion were induced by thermal stress, IL-6, or the IL-6/soluble IL-6 receptor fusion protein, hyper-IL-6. Analysis of the molecular basis of thermal regulation of L-selectin-like adhesion identified a common IL-6 trans-signaling mechanism in endotherms and ectotherms that resulted in activation of JAK/STAT signaling and was inhibited by IL-6 neutralizing antibodies or recombinant soluble gp130. Conservation of IL-6-dependent mechanisms controlling L-selectin adhesion over hundreds of millions of years of vertebrate evolution strongly suggests that this is a beneficial focal point regulating immune surveillance during febrile inflammatory responses.

[What is a Targeted Therapy? The View of the Biologist]

Bulletin Du Cancer. 2007  |  Pubmed ID: 17964987

Cancer chemotherapy, which followed a continuous progression all along the second half of the 20th century, is now undergoing profound changes: instead of focusing exclusively on the inhibition of cell proliferation, new developments now focus the mechanisms of oncogenesis. The name of "targeted therapy" refers to the targeting of oncogenesis. Understanding the mechanisms of oncogenesis is a requirement for the understanding of the use of targeted therapies. Each tumour follows its own natural history and characteristic oncogenic alterations, which define the potential targets for novel molecules. Prescribing targeted chemotherapy cannot be carried out without prior characterisation of the tumour, with the identification, among the various oncogenic alterations present, of the alteration to be targeted. As a consequence, it is mandatory to conceive this novel chemotherapy only on the basis of the selection of the patients who would benefit of it. This didactic review presents the general aspects of the molecular alterations of cancers, the potential targets which ensue, and the ways presently available to reach them.

Localization and Differential Expression of Activation-induced Cytidine Deaminase in the Amphibian Xenopus Upon Antigen Stimulation and During Early Development

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2007  |  Pubmed ID: 17982068

As in mammals, B cell maturation in the amphibian Xenopus involves somatic hypermutation (SHM) and class switch recombination to diversify the B cell receptor repertoire in response to Ag stimulation. Unlike mammals, however, the resulting increase in Ab affinity is poor in Xenopus, which is possibly related to the absence of germinal centers and a suboptimal selection mechanism of SHM. In mammals, both SHM and class switch recombination are mediated by the activation-induced cytidine deaminase enzyme and under Ag-dependent regulation. Given its evolutionary conservation in jawed vertebrates, we used activation-induced cytidine deaminase as a marker to monitor and localize B cell maturation in Xenopus upon immune responses and during early development. In adult, Xenopus laevis AID (XlAID) was detected mainly in the spleen, where cells expressing XlAID were preferentially distributed in follicular B cell zones, although some XlAID(+) cells were also found in the red pulp. XlAID was markedly up-regulated in the spleen with different kinetics upon bacterial stimulation and viral infection. However, during secondary anti-viral response XlAID was also noticeably expressed by PBLs, suggesting that XlAID remains active in a subset of circulating B cells. During ontogeny, XlAID expression was detected as early as 5 days postfertilization in liver before the first fully differentiated B cells appear. Concomitant with appearance of mature B cells XlAID was up-regulated upon bacterial stimulation or viral infection at later larval stages. This study highlights the conserved involvement of XlAID during Ag-dependent B cell responses in Xenopus but also suggests another role in B cell differentiation earlier in ontogeny.

Xenopus Laevis: a Possible Vector of Ranavirus Infection?

Journal of Wildlife Diseases. Oct, 2007  |  Pubmed ID: 17984259

Frog virus 3 (FV3) or FV3-like viruses (Iridoviridae) infect a wide range of amphibian species, and they are becoming increasingly and causally associated with amphibian disease outbreaks worldwide. We have established the frog Xenopus laevis as an experimental model to study host defense and pathogenesis of FV3 infection. Although X. laevis adults usually clear FV3 infection within a few weeks, viral DNA has been detected in the kidneys several months after they had been experimentally infected; virus also has been detected in seemingly healthy nonexperimentally infected adults. Based on this information, we hypothesized that covert FV3 infection may occur in Xenopus. We first conducted a survey that detected FV3 by polymerase chain reaction (PCR) in the kidneys (the main site of FV3 infection) in a significant fraction of X. laevis raised in five different locations in the United States. Asymptomatic FV3 carriers also were detected by initiation of an acute systemic FV3 infection in frogs that had been immunosupressed by sublethal gamma-irradiation. Finally, we focused on macrophages as a potential site for viral persistence, and we showed that FV3 can infect peritoneal macrophages in vitro as determined by reverse transcriptase-PCR detection of viral mRNAs. Unlike kidney cell lines that are readily killed by FV3, infected macrophages, like uninfected macrophages, survived up to 12 days. Viral transcription also was detected in macrophages from animals up to 12 days after infection. These results suggest that FV3 can become quiescent in resistant species such as Xenopus, thereby making these species potential viral reservoirs.

Determination of ERCC2 Lys751Gln and GSTP1 Ile105Val Gene Polymorphisms in Colorectal Cancer Patients: Relationships with Treatment Outcome

Pharmacogenomics. Dec, 2007  |  Pubmed ID: 18085999

Glutathione S-transferase P1 (GSTP1) and excision-repair cross-complementing repair deficiency group 2 protein (ERCC2 or XPD) may modulate the activity of platinum derivatives. The SNPs, Ile105Val for GSTP1 and Lys751Gln for ERCC2, may affect the efficiency of oxaliplatin in patients treated with an oxaliplatin-based regimen for metastatic colorectal carcinoma.

Phylogenetic Conservation of Glycoprotein 96 Ability to Interact with CD91 and Facilitate Antigen Cross-presentation

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2008  |  Pubmed ID: 18292541

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand alpha2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.

A Chemotherapy-associated Senescence Bystander Effect in Breast Cancer Cells

Cancer Biology & Therapy. Jun, 2008  |  Pubmed ID: 18340112

A bystander effect typically refers to the death, altered growth or damage of cells that have not directly received chemotherapy or irradiation. Cancer cells derived from solid tumors readily undergo senescence in response to chemotherapeutic agents, prompting us to test for the existence of a senescence bystander effect. MCF-7 breast cancer cells were acutely exposed to Adriamycin to trigger senescence. Naïve MCF-7 cells, when cultured in conditioned media from senescent breast cancer cells, growth arrested despite mitogenic stimulation and exhibited SA-beta-galactosidase activity, an enlarged cell size and stable upregulation of p21(WAF1) protein, collectively indicating a senescent state. In contrast, HCT-116 colon cancer cells, which also undergo p53-mediated senescence in response to acute AdR, did not undergo growth inhibition or senescence when cultured with conditioned media from senescent HCT-116 cells. Reciprocal experiments indicated that naïve HCT-116 cells, like MCF-7 cells, are susceptible to the growth inhibitory effects of a breast cancer-derived mediator, which is independent of residual drug in conditioned media. Our study reveals a novel action of Adriamycin, which may contribute to its potent anti-breast cancer activity and lead to the discovery of additional therapeutic targets for the exploitation of a senescence bystander effect.

Protein Arginine (N)-methyl Transferase 7 (PRMT7) As a Potential Target for the Sensitization of Tumor Cells to Camptothecins

FEBS Letters. Apr, 2008  |  Pubmed ID: 18381071

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

In Vivo and in Vitro Techniques for Comparative Study of Antiviral T-cell Responses in the Amphibian Xenopus

Biological Procedures Online. 2008  |  Pubmed ID: 18385804

Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogen-induced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a well-known amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.

The Xenopus FcR Family Demonstrates Continually High Diversification of Paired Receptors in Vertebrate Evolution

BMC Evolutionary Biology. 2008  |  Pubmed ID: 18485190

Recent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis.

Impact of EGFR Gene Polymorphisms on Anticancer Drug Cytotoxicity in Vitro

Molecular Diagnosis & Therapy. 2008  |  Pubmed ID: 18652519

The epidermal growth factor receptor (EGFR) plays a major role in cell proliferation of epithelial tissues, and its alterations frequently contribute to oncogenesis. Several common polymorphisms of the EGFR gene have been described, at the level of both coding and regulatory sequences. Some of these polymorphisms are associated with alterations of EGFR expression and/or activity and may have an impact on the activity of anticancer agents. This study aims to analyze the relationships between specific EGFR functional polymorphisms and anticancer drug activity.

[Biological Bases for Individualising Prescriptions in Oncology: the Germline Genome]

Bulletin Du Cancer. Oct, 2008  |  Pubmed ID: 19004720

Sequencing the human genome brings new tools for the individualisation of cancer chemotherapy, especially thanks to the identification of polymorphisms of genes involved in anticancer drug metabolism or activity (pharmacogenetics). A few functional polymorphisms have been known for a long time (thiopurine methyltransferase, glutathion S-transferases), but several new ones have been identified recently, at the level of the genes encoding drug targets (thymidylate synthase), at the level of DNA repair enzymes (XPD) or at the level of transport proteins (MDR1). Clinical trials, first on a retrospective basis, then on a prospective one, are implemented to validate this approach.

Synthesis and Antiproliferative Activity of Aryl- and Heteroaryl-hydrazones Derived from Xanthone Carbaldehydes

European Journal of Medicinal Chemistry. Jun, 2008  |  Pubmed ID: 17949859

In order to explore the antiproliferative effect associated with the xanthone framework, several arylhydrazonomethyl derivatives were synthesized from various isomeric 1,3-dihydroxyxanthone carbaldehydes. Variation in the position of the aldehydic function led to three sets of compounds, bearing the hydrazonomethyl chain at positions 5, 6 or 7 on the xanthone nucleus, respectively. The antiproliferative effect of the compounds was evaluated in vitro using the MTT colorimetric method against two human cancer cell lines (MCF-7, breast adenocarcinoma, and KB 3.1, squamous cell oral carcinoma) for two time periods (24 h and 72 h). Among the series, four compounds exhibited interesting growth inhibitory effects against both the cell lines, with IC(50) values in the micromolar concentration range. When compared with doxorubicin, the xanthone derivatives showed moderate cytotoxic effects. Surprisingly, unlike doxorubicin, these compounds displayed no significant time-dependent change in the concentration causing 50% inhibitory effect in proliferation. This unusual cytotoxicity profile led to the hypothesis that these molecules could be endowed with a mechanism of action distinct to that of doxorubicin.

Postmating Changes in Cuticular Chemistry and Visual Appearance in Ectatomma Tuberculatum Queens (Formicidae: Ectatomminae)

Die Naturwissenschaften. Jan, 2008  |  Pubmed ID: 17724573

In the ectatommine ant Ectatomma tuberculatum, the visual appearance of queens changes after mating and ovarian development in that their cuticle turns from shiny to matte. In this study, we have shown that this change seems to be caused by 15-fold accumulation of hydrocarbons, in particular heptacosane that covers the multiple grooves present on the cuticular surface creating a wax coat in mated fully fertile queens. Analyses of the scrapped wax revealed that it is composed largely of heptacosane. Peak-by-peak comparison of the cuticular hydrocarbon (CHC) composition of mated, virgin with developed ovaries and virgin with nondeveloped ovaries revealed significant differences between the queen groups. Although the total amount of the CHC of virgin queens with developed ovaries was not higher than virgin queens that did not have developed ovaries, the composition showed a shift toward the mated queen. While it is possible that the large accumulation of hydrocarbons may give extra physical and chemical protection to queens, we propose that the switch in the relative abundance of heptacosane and nonacosane and perhaps of other components is indicative of being a mating and fertility cue. This is the first report in social insects where external chemical changes are accompanied by changes in visual appearance.

Functional Study of the 830C>G Polymorphism of the Human Carboxylesterase 2 Gene

Cancer Chemotherapy and Pharmacology. Mar, 2008  |  Pubmed ID: 17483951

Carboxylesterase 2 (CES2) is involved in the activation of the anticancer drug irinotecan to its active metabolite SN-38. We previously identified a single nucleotide polymorphism (SNP), with an allele frequency around 10%, as possibly involved in enzyme expression (Clin Pharmacol Ther 76:528-535, 2004), which could explain the large individual variation in SN-38 disposition.

Genome-wide Transcriptional Response of Silurana (Xenopus) Tropicalis to Infection with the Deadly Chytrid Fungus

PloS One. 2009  |  Pubmed ID: 19701481

Emerging infectious diseases are of great concern for both wildlife and humans. Several highly virulent fungal pathogens have recently been discovered in natural populations, highlighting the need for a better understanding of fungal-vertebrate host-pathogen interactions. Because most fungal pathogens are not fatal in the absence of other predisposing conditions, host-pathogen dynamics for deadly fungal pathogens are of particular interest. The chytrid fungus Batrachochytrium dendrobatidis (hereafter Bd) infects hundreds of species of frogs in the wild. It is found worldwide and is a significant contributor to the current global amphibian decline. However, the mechanism by which Bd causes death in amphibians, and the response of the host to Bd infection, remain largely unknown. Here we use whole-genome microarrays to monitor the transcriptional responses to Bd infection in the model frog species, Silurana (Xenopus) tropicalis, which is susceptible to chytridiomycosis. To elucidate the immune response to Bd and evaluate the physiological effects of chytridiomycosis, we measured gene expression changes in several tissues (liver, skin, spleen) following exposure to Bd. We detected a strong transcriptional response for genes involved in physiological processes that can help explain some clinical symptoms of chytridiomycosis at the organismal level. However, we detected surprisingly little evidence of an immune response to Bd exposure, suggesting that this susceptible species may not be mounting efficient innate and adaptive immune responses against Bd. The weak immune response may be partially explained by the thermal conditions of the experiment, which were optimal for Bd growth. However, many immune genes exhibited decreased expression in Bd-exposed frogs compared to control frogs, suggesting a more complex effect of Bd on the immune system than simple temperature-mediated immune suppression. This study generates important baseline data for ongoing efforts to understand differences in response to Bd between susceptible and resistant frog species and the effects of chytridiomycosis in natural populations.

Expression Profiling the Temperature-dependent Amphibian Response to Infection by Batrachochytrium Dendrobatidis

PloS One. 2009  |  Pubmed ID: 20027316

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.

Mass Mortality Associated with a Frog Virus 3-like Ranavirus Infection in Farmed Tadpoles Rana Catesbeiana from Brazil

Diseases of Aquatic Organisms. Nov, 2009  |  Pubmed ID: 20066953

Ranaviruses (Iridoviridae) are increasingly associated with mortality events in amphibians, fish, and reptiles. They have been recently associated with mass mortality events in Brazilian farmed tadpoles of the American bullfrog Rana catesbeiana Shaw, 1802. The objectives of the present study were to further characterize the virus isolated from sick R. catesbeiana tadpoles and confirm the etiology in these outbreaks. Sick tadpoles were collected in 3 farms located in Goiás State, Brazil, from 2003 to 2005 and processed for virus isolation and characterization, microbiology, histopathology, and parasitology. The phylogenetic relationships of Rana catesbeiana ranavirus (RCV-BR) with other genus members was investigated by PCR with primers specific for the major capsid protein gene (MCP) and the RNA polymerase DNA-dependent gene (Pol II). Sequence analysis and multiple alignments for MCP products showed >99% amino acid identity with other ranaviruses, while Pol II products showed 100% identity. Further diagnostics of the pathology including histology and transmission electron microscopy confirmed the viral etiology of these mass deaths. As far as we know, this is the first report of a ranaviral infection affecting aquatic organisms in Brazil. Additionally, our results suggest that American bullfrogs may have served as a vector of transmission of this virus, which highlights the potential threat of amphibian translocation in the world distribution of pathogens.

Patient Geometry-driven Information Retrieval for IMRT Treatment Plan Quality Control

Medical Physics. Dec, 2009  |  Pubmed ID: 20095262

Intensity modulated radiation therapy (IMRT) treatment plan quality depends on the planner's level of experience and the amount of time the planner invests in developing the plan. Planners often unwittingly accept plans when further sparing of the organs at risk (OARs) is possible. The authors propose a method of IMRT treatment plan quality control that helps planners to evaluate the doses of the OARs upon completion of a new plan.

A Shape Relationship Descriptor for Radiation Therapy Planning

Medical Image Computing and Computer-assisted Intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention. 2009  |  Pubmed ID: 20426101

In this paper we address the challenge of matching patient geometry to facilitate the design of patient treatment plans in radiotherapy. To this end we propose a novel shape descriptor, the Overlap Volume Histogram, which provides a rotation and translation invariant representation of a patient's organs at risk relative to the tumor volume. Using our descriptor, it is possible to accurately identify database patients with similar constellations of organ and tumor geometries, enabling the transfer of treatment plans between patients with similar geometries, We demonstrate the utility of our method for such tasks by outperforming state of the art shape descriptors in the retrieval of patients with similar treatment plans. We also preliminarily show its potential as a quality control tool by demonstrating how it is used to identify an organ at risk whose dose can be significantly reduced.

Xenopus, a Unique Comparative Model to Explore the Role of Certain Heat Shock Proteins and Non-classical MHC Class Ib Gene Products in Immune Surveillance

Immunologic Research. Feb, 2009  |  Pubmed ID: 19189057

The heat shock proteins (HSPs) gp96 and hsp70 can elicit potent anti-tumor responses and as such have significant clinical potential. Besides cytotoxic CD8 T cell (CTLs) effectors, evidence suggests that natural killer (NK) cells and other less well-characterized cell types also play a critical role in HSP-mediated anti-tumor responses. Owing to their high degree of phylogenetic conservation, we have proposed that HSPs are ancestral agents of immune surveillance; and postulated that their immunological properties, if important, should have been conserved during evolution. We are investigating this issue using a unique non-mammalian comparative tumor-immunity model in the frog Xenopus, which allows us to focus on the relationship between HSPs, classical MHC class Ia, and non-classical MHC class Ib molecules. In addition to a transplantable lymphoid tumor in genetically defined cloned Xenopus, we are generating transgenic frogs with inducible or knocked-down (RNAi) gene expression.

Novel Nonclassical MHC Class Ib Genes Associated with CD8 T Cell Development and Thymic Tumors

Molecular Immunology. May, 2009  |  Pubmed ID: 19237199

In jawed vertebrates, the heterogeneous nonclassical MHC class Ib (class Ib) gene family encodes molecules structurally similar to classical MHC class Ia (class Ia) but with more limited tissue distribution and lower polymorphism. In mammals, class Ib gene products are involved in stress responses, malignancy and differentiation of intrathymic CD8 T cells. The frog Xenopus laevis possesses at least 20 class Ib genes (XNCs), and 9 subfamilies have been defined so far. We have characterized two novel subfamilies, XNC10 and XNC11. XNC10 is phylogenetically and structurally distinct from both class Ia and other XNC genes. Besides thymic lymphoid tumors, XNC10 is preferentially expressed by circulating T cells and thymocytes of the CD8 lineage both in adult and in larvae from the onset of thymus organogenesis. XNC11 is expressed only by thymocytes and upregulated by several thymic lymphoid tumors. These data provide the first evidence of the expression of any class Ib genes in Xenopus larvae, and suggests evolutionary relationships between certain class Ib genes, malignancy and CD8 T cell ontogeny.

Comparative and Developmental Study of the Immune System in Xenopus

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2009  |  Pubmed ID: 19253402

Xenopus laevis is the model of choice for evolutionary, comparative, and developmental studies of immunity, and invaluable research tools including MHC-defined clones, inbred strains, cell lines, and monoclonal antibodies are available for these studies. Recent efforts to use Silurana (Xenopus) tropicalis for genetic analyses have led to the sequencing of the whole genome. Ongoing genome mapping and mutagenesis studies will provide a new dimension to the study of immunity. Here we review what is known about the immune system of X. laevis integrated with available genomic information from S. tropicalis. This review provides compelling evidence for the high degree of similarity and evolutionary conservation between Xenopus and mammalian immune systems. We propose to build a powerful and innovative comparative biomedical model based on modern genetic technologies that takes take advantage of X. laevis and S. tropicalis, as well as the whole Xenopus genus. Developmental Dynamics 238:1249-1270, 2009. (c) 2009 Wiley-Liss, Inc.

Diversity of the FcR- and KIR-related Genes in an Amphibian Xenopus

Frontiers in Bioscience : a Journal and Virtual Library. 2009  |  Pubmed ID: 19273057

Receptors subdivided into inhibitory and activating forms play important roles in the regulation of leukocyte development and effector functions. Two prototypic examples of paired receptors are Fc-receptors (FcR) and Killer cell Immunoglobulin-like receptors (KIR). FcRs are cell surface proteins that bind to the constant regions of IgG and IgE. Classical KIRs recognize MHC class I molecules and regulate natural killer (NK) cell cytotoxic functions. The evolution of these proteins and the time of their origin remain enigmatic. So far, molecules unequivocally related to mammalian FcRs and KIRs have been identified in chicken and an amphibian Xenopus. The lineage-specific evolution of the FcR and KIR families apparently led to the generation of unique sets of receptors in all species studied. Members of both families show extraordinary diversity of domain architectures. This structural diversity makes elusive the functional relationships between the highly specialized mammalian FcR and KIR genes and their homologs in nonmammalian species.

Tumorigenesis and Anti-tumor Immune Responses in Xenopus

Frontiers in Bioscience : a Journal and Virtual Library. 2009  |  Pubmed ID: 19273061

Despite intense study, the role of the immune system in detecting (immunosurveillance), controlling and remodeling (immunoediting) neoplasia remains elusive. We present here a comparative view of the complex interactions between neoplasia and the host immune system. We provide evidence, in the amphibian Xenopus laevis, consistent with an evolutionarily conserved and crucial role of the immune system in controlling neoplasia, which involves a striking variety of anti-tumoral immune effectors including conventional CTLs, classical MHC class Ia unrestricted CTLs (CCU-CTLs) that interact with nonclassical MHC class Ib molecules, CD8 NKT-like cells and NK cells. We also review the tumors found in X. laevis with an emphasis on thymic lymphoid tumors and a rare ovarian dysgerminoma. Finally, we consider the use of X. laevis for in vivo study of tumorigenesis. Given our current knowledge, the experimental systems already established in X. laevis, and the rapid accumulation of genetic resources for the sister species Silurana (Xenopus) tropicalis, it is our conviction that these species provide an ideal alternative to the murine system for studying tumorigenesis and tumor immunity.

Control of Asthma in Children: Still Unacceptable? A French Cross-sectional Study

Respiratory Medicine. Sep, 2009  |  Pubmed ID: 19361973

The goal of asthma management focuses on adequate control of asthma, although little is known about the optimal level of asthma control to be reached. The ELIOS study was conducted in France to address this lack of information.

Isolation of a Latimeria Menadoensis Heat Shock Protein 70 (Lmhsp70) That Has All the Features of an Inducible Gene and Encodes a Functional Molecular Chaperone

Molecular Genetics and Genomics : MGG. Aug, 2009  |  Pubmed ID: 19444470

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

Involvement of Gene Polymorphisms of the Folate Pathway Enzymes in Gene Expression and Anticancer Drug Sensitivity Using the NCI-60 Panel As a Model

European Journal of Cancer (Oxford, England : 1990). Sep, 2009  |  Pubmed ID: 19501504

Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N(5,10)-methylene-tetrahydrofolate reductase), MTHFD1 (N(5,10)-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity.

Towards Real-time Radiation Therapy: GPU Accelerated Superposition/convolution

Computer Methods and Programs in Biomedicine. Jun, 2010  |  Pubmed ID: 19695731

We demonstrate the use of highly parallel graphics processing units (GPUs) to accelerate the superposition/convolution (S/C) algorithm to interactive rates while reducing the number of approximations. S/C first transports the incident fluence to compute the total energy released per unit mass (TERMA) grid. Dose is then calculated by superimposing the dose deposition kernel at each point in the TERMA grid and summing the contributions to the surrounding voxels. The TERMA algorithm was enhanced with physically correct multi-spectral attenuation and a novel inverse formulation for increased performance, accuracy and simplicity. Dose deposition utilized a tilted poly-energetic inverse cumulative-cumulative kernel, with the novel option of using volumetric mip-maps to approximate solid angle ray casting. Exact radiological path ray casting decreased discretization errors. We achieved a speedup of 34x-98x over a highly optimized CPU implementation.

Landau-Zener Transitions in Frozen Pairs of Rydberg Atoms

Physical Review Letters. Apr, 2010  |  Pubmed ID: 20481882

We have induced adiabatic transitions in pairs of frozen Rydberg sodium atoms of a supersonic beam. The diatomic ns+ns-->np+(n-1)p transition takes place in a time-dependent electric field and originates from the adiabatic change of the internal state of the pair induced by the dipole-dipole interaction. This is experimentally achieved by sweeping an electric field across the energy degeneracy ns ns-np(n-1)p. Our results fully agree with a two-level Landau-Zener model in the diatom system.

Comparative Study of Tumorigenesis and Tumor Immunity in Invertebrates and Nonmammalian Vertebrates

Developmental and Comparative Immunology. Sep, 2010  |  Pubmed ID: 20553753

Despite intense study in mammals, the different roles played by the immune system in detecting (immunosurveillance), controlling and remodeling (immunoediting) neoplasia, and perhaps in metastasis are not fully understood. In this review, I will present evidence of neoplasia and invasive malignancy, as well as tumor immunity in invertebrates and nonmammalian vertebrates. I will also present a comparative and evolutionary view of the complex interactions between neoplasia and the host immune system. Overall, I wish to go beyond the too simplistic dichotomy between invertebrates with innate immunity that are only affected with benign neoplasia and vertebrates with adaptive immunity that are affected by metastatic malignancies or cancer.

Cytochrome P450 1B1 Gene Polymorphisms As Predictors of Anticancer Drug Activity: Studies with in Vitro Models

Molecular Cancer Therapeutics. Dec, 2010  |  Pubmed ID: 20889730

Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.

Recommended Nomenclature for Five Mammalian Carboxylesterase Gene Families: Human, Mouse, and Rat Genes and Proteins

Mammalian Genome : Official Journal of the International Mammalian Genome Society. Oct, 2010  |  Pubmed ID: 20931200

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

The Genome of the Western Clawed Frog Xenopus Tropicalis

Science (New York, N.Y.). Apr, 2010  |  Pubmed ID: 20431018

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

A Statistical Approach for Achievable Dose Querying in IMRT Planning

Medical Image Computing and Computer-assisted Intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention. 2010  |  Pubmed ID: 20879440

The task of IMRT planning, particularly in head-and-neck cancer, is a difficult one, often requiring days of work from a trained dosimetrist. One of the main challenges is the prescription of achievable target doses that will be used to optimize a treatment plan. This work explores a data-driven approach in which effort spent on past plans is used to assist in the planning of new patients. Using a database of treated patients, we identify the features of patient geometry that are correlated with received dose and use these to prescribe target dose levels for new patients. We incorporate our approach in a quality-control system, identifying patients with organs that received a dose significantly higher than the one recommended by our method. For all these patients, we have found that a replan using our predicted dose results in noticeable sparing of the organ without compromising dose to other treatment volumes.

Innate Immune Responses and Permissiveness to Ranavirus Infection of Peritoneal Leukocytes in the Frog Xenopus Laevis

Journal of Virology. May, 2010  |  Pubmed ID: 20200236

Ranaviruses such as frog virus 3 ([FV3] family Iridoviridae) are increasingly prevalent pathogens that infect reptiles, amphibians, and fish worldwide. Whereas studies in the frog Xenopus laevis have revealed the critical involvement of CD8 T-cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry, and reverse transcription-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day postinfection (dpi), followed by NK cells at 3 dpi, before the peak of the T-cell response at 6 dpi. FV3 infection also induces a rapid upregulation of proinflammatory genes including arginase 1, interleukin-1beta, and tumor necrosis factor alpha. Although PLs are susceptible to FV3 infection, as evidenced by apoptotic cells, active FV3 transcription, and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory, and involves a smaller fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks postinfection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

Phylogeny, Genomic Organization and Expression of Lambda and Kappa Immunoglobulin Light Chain Genes in a Reptile, Anolis Carolinensis

Developmental and Comparative Immunology. May, 2010  |  Pubmed ID: 20056120

The reptiles are the last major taxon of jawed vertebrates in which immunoglobulin light chain isotypes have not been well characterized. Using the recently released genome sequencing data, we show in this study that the reptile Anolis carolinensis expresses both lambda and kappa light chain genes. The genomic organization of both gene loci is structurally similar to their respective counterparts in mammals. The identified lambda locus contains three constant region genes each preceded by a joining gene segment, and a total of 37 variable gene segments. In contrast, the kappa locus contains only a single constant region gene, and two joining gene segments with a single family of 14 variable gene segments located upstream. Analysis of junctions of the recombined VJ transcripts reveals a paucity of N and P nucleotides in both expressed lambda and kappa sequences. These results help us to understand the generation of the immunoglobulin repertoire in reptiles and immunoglobulin evolution in vertebrates.

The Amphibians Xenopus Laevis and Silurana Tropicalis Possess a Family of Activating KIR-related Immunoglobulin-like Receptors

Developmental and Comparative Immunology. Mar, 2010  |  Pubmed ID: 19896971

In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

Optimized Transgenesis in Xenopus Laevis/gilli Isogenetic Clones for Immunological Studies

Genesis (New York, N.Y. : 2000). Sep, 2011  |  Pubmed ID: 21954010

Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the X. laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase, and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35-50%), bright nonmosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen X. laevis as a biomedical model. genesis 00:1-7, 2011. © 2011 Wiley Periodicals, Inc.

[Tyrosine Kinase Inhibitors]

Bulletin Du Cancer. Nov, 2011  |  Pubmed ID: 22023787

Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

Antiviral Immunity in Amphibians

Viruses. Nov, 2011  |  Pubmed ID: 22163335

Although a variety of virus species can infect amphibians, diseases caused by ranaviruses ([RVs]; Iridoviridae) have become prominent, and are a major concern for biodiversity, agriculture and international trade. The relatively recent and rapid increase in prevalence of RV infections, the wide range of host species infected by RVs, the variability in host resistance among population of the same species and among different developmental stages, all suggest an important involvement of the amphibian immune system. Nevertheless, the roles of the immune system in the etiology of viral diseases in amphibians are still poorly investigated. We review here the current knowledge of antiviral immunity in amphibians, focusing on model species such as the frog Xenopus and the salamander (Ambystoma tigrinum), and on recent progress in generating tools to better understand how host immune defenses control RV infections, pathogenicity, and transmission.

Phylogenetic and Developmental Study of CD4, CD8 α and β T Cell Co-receptor Homologs in Two Amphibian Species, Xenopus Tropicalis and Xenopus Laevis

Developmental and Comparative Immunology. Mar, 2011  |  Pubmed ID: 21075137

CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8β gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species Xenopus laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the co-evolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8β expression is specific to adult surface CD8(+) T cells and thymocytes, and is first detected in the thymus at 5 dpf in parallel with productive TCRγ transrcipts, whereas productive TCRβ and α rearrangements are not detected before 7-9 dpf.

ERCC5/XPG, ERCC1, and BRCA1 Gene Status and Clinical Benefit of Trabectedin in Patients with Soft Tissue Sarcoma

Cancer. Aug, 2011  |  Pubmed ID: 21287534

The objective of this study was to determine whether specific single nucleotide polymorphisms (SNPs) from nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways are associated with sensitivity to trabectedin in patients with soft tissue sarcoma (STS).

Real-time Dose Computation: GPU-accelerated Source Modeling and Superposition/convolution

Medical Physics. Jan, 2011  |  Pubmed ID: 21361198

To accelerate dose calculation to interactive rates using highly parallel graphics processing units (GPUs).

[General Overview on DNA Repair]

Bulletin Du Cancer. Mar, 2011  |  Pubmed ID: 21414892

DNA repair is implemented through a large variety of mechanisms, each of them being adapted to a specific type of lesion: direct repair, mismatch repair for the errors occurring during the replication process, base-excision repair, nucleotide-excision repair, double-strand breaks DNA repair by homologous or non-homologous recombination. Each of these mechanisms involves numerous proteins associated as supramolecular functional complexes. Some anticancer drugs are able to generate DNA lesions which may overflow the repair mechanisms. The impossibility to repair DNA damage usually leads to cell death, but alterations of repair mechanisms may favour genetic instability and hence contribute to oncogenesis.

Role of DNA Repair Gene Polymorphisms in the Efficiency of Platinum-based Adjuvant Chemotherapy for Non-small Cell Lung Cancer

Molecular Diagnosis & Therapy. Jun, 2011  |  Pubmed ID: 21766907

Cisplatin-based adjuvant treatment of non-small cell lung cancer (NSCLC) has become standard, thanks to the studies that have shown a significant survival advantage. The identification of patients who could benefit from this adjuvant treatment would allow ineffective and toxic administrations to be avoided. Immunohistochemical expression of the excision repair cross-complementation group (ERCC)-1 protein has been associated with response to platinum-based chemotherapy in patients with NSCLC, and some polymorphisms of the genes involved in DNA repair have been shown to be associated with survival in advanced NSCLC.

Waterborne Infectivity of the Ranavirus Frog Virus 3 in Xenopus Laevis

Virology. Sep, 2011  |  Pubmed ID: 21783222

Ranaviruses like frog virus 3 (FV3) are responsible for emerging infectious diseases spreading worldwide to fish, amphibian and reptilian species. We have developed, in Xenopus laevis, an experimental model to investigate viral transmission. We show that FV3 released in water by immunocompromised infected adults can infect adult and larval stages of Xenopus within 3h of exposure. Time course of virus load and viral transcription in different tissues suggests that early waterborne FV3 infection through the digestive tract leads to dissemination in the kidney. Finally, a fraction of adult macrophages becomes infected following exposure to waterborne FV3 as visualized by fluorescence microscopy using macrophage- and FV3-specific antibodies. Little cytopathicity and apoptosis were detected in infected macrophages, which is consistent with our proposition that macrophages are permissive to FV3. These data highlight the efficiency of FV3 infectivity by the water route and the ability of FV3 to adapt to its hosts.

Encapsulation of Docetaxel into PEGylated Gold Nanoparticles for Vectorization to Cancer Cells

ChemMedChem. Nov, 2011  |  Pubmed ID: 21834092

Encapsulation of docetaxel and its solubilization in water was carried out in PEGylated gold nanoparticles (AuNPs) as shown by 1H NMR (600 MHz) and UV/Vis spectroscopy and dynamic light scattering. Vectorization of PEGylated AuNP-encapsulated docetaxel was probed in vitro toward human colon carcinoma (HCT15) and human breast cancer (MCF7) cells. AuNPs alone presented no cytotoxicity toward either MCF7 or HCT15 adenocarcinoma cells. AuNP-docetaxel was found to be 2.5-fold more efficient than docetaxel alone against MCF7 cells, and the IC50 value of AuNP-docetaxel against HCT15 cells was lower than that of free docetaxel; the increased efficiency brought about by AuNP drug encapsulation was ∼1.5-fold.

Improved Knockout Methodology Reveals That Frog Virus 3 Mutants Lacking Either the 18K Immediate-early Gene or the Truncated VIF-2alpha Gene Are Defective for Replication and Growth in Vivo

Journal of Virology. Nov, 2011  |  Pubmed ID: 21865381

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.

Data-driven Approach to Generating Achievable Dose-volume Histogram Objectives in Intensity-modulated Radiotherapy Planning

International Journal of Radiation Oncology, Biology, Physics. Mar, 2011  |  Pubmed ID: 20800382

To propose a method of intensity-modulated radiotherapy (IMRT) planning that generates achievable dose-volume histogram (DVH) objectives using a database containing geometric and dosimetric information of previous patients.

Remarkable Conservation of Distinct Nonclassical MHC Class I Lineages in Divergent Amphibian Species

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2011  |  Pubmed ID: 21115732

Nonclassical MHC class Ib (class Ib) genes are heterogeneous genes encoding molecules that are structurally similar to classical MHC class Ia molecules but with limited tissue distribution and polymorphism. Mammalian class Ib genes have diverse and often uncharacterized functions, and because of their rapid rate of evolution, class Ib phylogeny is difficult to establish. We have conducted an extensive genomic, molecular, and phylogenetic characterization of class Ib genes in two Xenopodinae amphibian species of different genera that diverged from a common ancestor as long ago as primates and rodents (∼65 million years). In contrast with the unsteadiness of mammalian class Ib genes, our results reveal an unusual degree of conservation of most Xenopodinae class Ib gene lineages, including a novel monogenic lineage represented by the divergent Xenopus laevis XNC10 gene and its unequivocal Silurana (Xenopus) tropicalis orthologue, SNC10. The preferential expression of this gene lineage by thymocytes themselves from the onset of thymic organogenesis is consistent with a specialized role of class Ib in early T cell development and suggests such a function is conserved in all tetrapods.

The Genus Xenopus As a Multispecies Model for Evolutionary and Comparative Immunobiology of the 21st Century

Developmental and Comparative Immunology. Sep, 2011  |  Pubmed ID: 21277325

The Xenopus model for immunological research offers a collection of invaluable research tools including MHC-defined clones, inbred strains, cell lines, and monoclonal antibodies. Further, the annotated full genome sequence of Xenopus tropicalis and its remarkable conservation of gene organization with mammals, as well as ongoing genome mapping and mutagenesis studies in X. tropicalis, add a new dimension to the study of immunity. In this paper, we review uses of this amphibian model to study: the development of the immune system; vascular and lymphatic regeneration; immune tolerance; tumor immunity; immune responses to important emerging infectious diseases; and the evolution of classical and non-classical MHC class I genes. We also discuss the rich potential of the species with different degrees of polypoidy resulting from whole genome-wide duplication of the Xenopodinae subfamily as a model to study regulation at the genome level.

ERCC1 and ERCC2 Polymorphisms Predict Clinical Outcomes of Oxaliplatin-based Chemotherapies in Gastric and Colorectal Cancer: a Systemic Review and Meta-analysis

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2011  |  Pubmed ID: 21278243

Nucleotide excision repair (NER) modulates platinum-based chemotherapeutic efficacy by removing drug-produced DNA damage. To summarize published data on the association between polymorphisms of NER genes (ERCC1 and ERCC2) and responses to oxaliplatin-based chemotherapies, we carried out a meta-analysis of gastric and colorectal cancer for commonly studied polymorphisms ERCC1 rs11615C>T and ERCC2 rs13181T>G.

"Ranaviruses: an Emerging Threat to Ectothermic Vertebrates" Report of the First International Symposium on Ranaviruses, Minneapolis MN July 8, 2011

Developmental and Comparative Immunology. Feb, 2012  |  Pubmed ID: 21906623

This is a report of the First International Symposium on Ranaviruses held on July 8, 2011 in conjunction with the annual Joint Meeting of Ichthyologists and Herpetologists (JMIH) in Minneapolis, Minnesota, USA. The emerging threat of ranavirus infectious diseases to the global biodiversity of ectothermic vertebrates was addressed by 23 scientists from nine countries with expertise in ecology, pathology, virology, veterinary medicine and immunology.

Absence of Transcriptomic Signature of Response to Chemotherapy in Metastatic Colorectal Carcinoma Patients

Pharmacogenomics. Feb, 2012  |  Pubmed ID: 22329722

Aim: Tumor gene-expression profiling may define signatures capable of discriminating between responders and nonresponders to chemotherapy. Patients & methods: Fifty seven metastatic colorectal cancer patients were prospectively included and 40 tumors were analyzed. Patients were treated in first line with 5-fluorouracil associated with irinotecan or oxaliplatin. Response was evaluated using WHO criteria every 2 months after chemotherapy. Gene-expression profiling was performed using Applied Biosystems microarrays (Human Genome Survey Microarray v2.0; Paris, France). Data were analyzed using Bioconductor packages. Differential-expression analysis was performed by fitting a linear model. Moderated t-statistics were computed and p-values were adjusted for false-discovery rate. Pearson correlations tests were evaluated between gene expression and progression-free and overall survival. Results: Nonsupervised analysis did not show any clustering of expression levels according to treatment response. Supervised analysis compared expression levels between responders and nonresponders, within each treatment group and independently from treatment. No genes were identified as differentially expressed at a p-value of 10(-3) and false-discovery rate of 30%. No correlation between expression levels and survival data was found. Conclusion: These negative results show that the determinants of response to chemotherapy should be sought not only in the tumor characteristics, but also among the processes leading to drug availability to the tumor. Original submitted 7 July 2011; Revision submitted 17 October 2011.

7β-Hydroxycholesterol-induced Energy Stress Leads to Sequential Opposing Signaling Responses and to Death of C6 Glioblastoma Cells

Biochemical Pharmacology. Jan, 2012  |  Pubmed ID: 21983033

7β-Hydroxycholesterol cytotoxicity has been shown in vivo and in vitro to be dependent on the accumulation of its esters. We show in our study, using a detergent-free raft preparation and LC/MS lipid content analysis, that membrane microdomains isolated from 7β-hydroxycholesterol-treated C6 cells have a reduced cholesterol: cholesterol ester ratio and accumulate 7keto-hydroxycholesterol, 7β-hydroxycholesterol and 7β-hydroxycholesterol esters. These modifications in lipid content are accompanied by a redistribution of flotillin-1 in the lipid rafts. Transient increases of AMPK phosphorylation and mitochondrial activity during the first 12 h of 7β-hydroxycholesterol treatment indicate that C6 cells undergo energy stress and increase oxidative phosphorylation. Even so, ATP levels are maintained during 15 h until glucose uptake decreases. The cell's answers to raft modifications and energy stress are sequential activations of different signaling pathways such as ERK, AMPK and PI3K/Akt. These pathways, known to be activated under energy stress conditions, are transiently activated at 6 h (ERK, AMPK) and 12 h (Akt) of treatment respectively suggesting a shift from cell survival to cell proliferation. The persistence of 7β-hydroxycholesterol-induced stress led after 24 h to P38 activation, loss of GSK3β activation and to cell death. Finally we demonstrate that the observed signaling responses depend on 7β-hydroxycholesterol esterification, confirming that esterification of 7β-hydroxycholesterol is essential for cytotoxicity.