RÉSUMÉ
Myeloblastosis-associated virus 2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in the kidneys, lungs, and liver with a short latency, less than 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHSs), intrahepatic cholangiocarcinomas (ICCs), and hepatocellular carcinomas (HCCs). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca. 86% of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON Insertionally mutated genes correlated with tumor type: HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs, and EGFR mostly in HCCs. The provirus insertions led to the overexpression of the affected genes and, in the case of EGFR and RON, also to the truncation of exons encoding the extracellular ligand-binding domains of these transmembrane receptors. The structures of truncated EGFR and RON closely mimic the structures of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).IMPORTANCE These data describe the mechanisms of oncogenesis induced in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.
Sujet(s)
Virus de la myéloblastose aviaire/physiologie , Carcinogenèse , Gènes erbB-1 , Tumeurs du foie/virologie , Mutagenèse par insertion , Protéines proto-oncogènes c-met/génétique , Récepteurs à activité tyrosine kinase/génétique , Animaux , Virus de la myéloblastose aviaire/génétique , Protéines aviaires/génétique , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/virologie , Poulets/génétique , Cholangiocarcinome/génétique , Cholangiocarcinome/virologie , Hémangiosarcome/génétique , Hémangiosarcome/virologie , Humains , Tumeurs du foie/génétique , Oncogènes , Provirus/génétique , Provirus/physiologie , Intégration viraleRÉSUMÉ
The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.
Sujet(s)
Protéines aviaires/métabolisme , Différenciation cellulaire , Mélanocytes/physiologie , Crête neurale/cytologie , Protéines oncogènes v-myb/physiologie , Activation de la transcription , Allèles , Animaux , Protéines aviaires/génétique , Protéine morphogénétique osseuse de type 5/génétique , Protéine morphogénétique osseuse de type 5/métabolisme , Cellules cultivées , Embryon de poulet , Régulation de l'expression des gènes , Protéines et peptides de signalisation intercellulaire/génétique , Protéines et peptides de signalisation intercellulaire/métabolisme , Kératine-19/génétique , Kératine-19/métabolisme , Séquençage par oligonucléotides en batterie , TranscriptomeRÉSUMÉ
Fibrotic diseases are a group of pathologies with high incidence and mortality. Despite extensive research efforts, effective therapies are still not available. Understanding the molecular mechanisms driving the onset, progression and possible resolution of fibrosis is a prerequisite to the development of successful therapies. The central role of the TGF-ß pathway and myofibroblasts in the pathogenesis of fibrosis is now generally accepted. The possible mechanisms of myofibroblast elimination or dedifferentiation, on the other hand, are still almost uncharted territory. Here we show that sustained expression of some components of MAPK signaling pathway (PDGFB, Ha-Ras(G12V) or the transcription factor EGR4) in primary chicken embryo dermal myofibroblasts results in a loss of autocrine TGF-ß signaling and suppression of the myofibroblastic phenotype, characterized by the loss of alpha smooth muscle actin fibers and a substantial reduction in the production of extracellular matrix. Detailed analysis of the possible molecular mechanisms employed by EGR4 revealed FOXG1, BAMBI, NAB1, NAB2 and DUSP5 genes forming an EGR4 regulated network counteracting autocrine TGF-ß signaling. We have also found that a combination of chemical inhibition of TGF-ß signaling and perturbation of MAPK signaling with phorbol ester mimics the anti-fibrotic effects of PDGFB, Ha-Ras(G12V) and EGR4.
Sujet(s)
Dédifférenciation cellulaire , Mitogen-Activated Protein Kinases/métabolisme , Myofibroblastes/métabolisme , Facteur de croissance transformant bêta/métabolisme , Actines/génétique , Actines/métabolisme , Animaux , Embryon de poulet , Myofibroblastes/cytologie , Esters de phorbol/pharmacologie , Transduction du signalRÉSUMÉ
BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.
Sujet(s)
Chaînes légères de myosine/métabolisme , Sarcomes/métabolisme , Protéines G rho/métabolisme , rho-Associated Kinases/métabolisme , Animaux , Lignée cellulaire tumorale , Mouvement cellulaire , Poulets , Invasion tumorale , Rats , Sarcomes/anatomopathologie , Transduction du signalRÉSUMÉ
The myofibroblast is a mesenchymal cell characterized by synthesis of the extracellular matrix, plus contractile and secretory activities. Myofibroblasts participate in physiological tissue repair, but can also cause devastating fibrosis. They are present in the tumor stroma of carcinomas and contribute to tumor growth and spreading. As myofibroblasts derive from various cell types and appear in a variety of tissues, there is marked variability in their phenotype. As regulatory mechanisms of wound healing are likely conserved among vertebrates, detailed knowledge of these mechanisms in more distant species will help to distinguish general from specific phenomena. To provide this as yet missing comparison, we analyzed the impact of the chemical inhibition of TGF-beta signaling on gene expression in chicken embryo dermal myofibroblasts. We revealed genes previously reported in mammalian systems (e.g. SPON2, ASPN, COMP, LUM, HAS2, IL6, CXCL12, VEGFA) as well as novel TGF-beta dependent genes, among them PGF, VEGFC, PTN, FAM180A, FIBIN, ZIC1, ADCY2, RET, HHIP and DNER. Inhibition of TGF-beta signaling also induced multiple genes, including NPR3, AGTR2, MTUS1, SOD3 and NOV. We also analyzed the effects of long term inhibition, and found that it is not able to induce myofibroblast dedifferentiation.
Sujet(s)
Poulets/génétique , Derme/embryologie , Régulation de l'expression des gènes au cours du développement , Myofibroblastes/physiologie , Facteur de croissance transformant bêta/métabolisme , Animaux , Cellules cultivées , Embryon de poulet , Derme/effets des médicaments et des substances chimiques , Myofibroblastes/effets des médicaments et des substances chimiques , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Récepteur de type II du facteur de croissance transformant bêta , Récepteurs TGF-bêta/antagonistes et inhibiteurs , Transduction du signal/effets des médicaments et des substances chimiques , Facteur de croissance transformant bêta/antagonistes et inhibiteursRÉSUMÉ
Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines-the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.
Sujet(s)
Transformation cellulaire néoplasique/génétique , Facteur de transcription EGR-1/métabolisme , Métastase tumorale/génétique , Protéine oncogène pp60(v-src)/métabolisme , Sarcomes/génétique , Sarcomes/anatomopathologie , Animaux , Adhérence cellulaire , Lignée cellulaire , Mouvement cellulaire , Prolifération cellulaire , Transformation cellulaire néoplasique/anatomopathologie , Poulets , Cytosquelette/métabolisme , Facteur de transcription EGR-1/génétique , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes tumoraux , Cinétique , Protéine oncogène pp60(v-src)/génétique , PhénotypeRÉSUMÉ
A tumor cell is formed when a critical amount of endogenous and/or exogenous tumorigenic stimuli is exceeded. We have shown that the transient presence of nontumorigenic stray cells in tissues of experimental animals that contain cells with a subcritical set of genetic mutations can act as a tumor-promoting stimulus. To induce somatic mutations in all chicken tissues, we have used the MAV-2 retroviral insertion system that almost exclusively generates nephroblastomas. MAV-2 mutagenized animals i.v. inoculated with nonmalignant cells developed early clonal lung tumors before nephroblastomas. Importantly, the injected cells did not become a component of resultant tumors. Lung tumors displayed specific mutational signature characterized by an insertion of MAV-2 provirus into the fyn-related kinase (frk) promoter that results in the overexpression of the frk gene. In contrast, plag1, foxP, and twist genes were most often mutagenized in nephroblastomas. Based on such observations, we propose the mechanism termed industasis, a promotion of fully malignant phenotype of incipient tumor cell by stray cells, and hypothesize that it might be the underlying cause of human multiple primary tumors.
Sujet(s)
Transformation cellulaire néoplasique/anatomopathologie , Cellules/anatomopathologie , Animaux , Mouvement cellulaire/physiologie , Cellules/virologie , Cellules cultivées , Embryon de poulet , Poulets , Tumeurs du rein/anatomopathologie , Tumeurs du rein/virologie , Tumeurs du poumon/anatomopathologie , Tumeurs du poumon/virologie , Modèles biologiques , Mutagenèse par insertion/physiologie , Invasion tumorale , Tumeurs primitives multiples/étiologie , Provirus/croissance et développement , Provirus/physiologie , Phénomènes physiologiques viraux , Tumeur de Wilms/anatomopathologie , Tumeur de Wilms/virologieRÉSUMÉ
FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML), with internal tandem duplications (ITDs) accounting for up to 30% of its mutations. To analyze the impact of individual ITDs on the expression profile of immature myeloid cells, we have established 32D cell lines expressing nine different FLT3/ITDs isolated from AML patients and subjected them to whole genome expression profiling and 2DE/LC/MS proteomics. Our data indicate that in comparison to the controls, FLT3/ITD-positive 32D cells exhibit less mature expression profiles resembling early hematopoietic progenitors. Moreover, our results suggest that there exist biological differences among individual ITD variants.
Sujet(s)
Crise blastique/génétique , Crise blastique/anatomopathologie , Variation génétique , Leucémie aigüe myéloïde/génétique , Séquences répétées en tandem/génétique , Tyrosine kinase-3 de type fms/génétique , Séquence d'acides aminés , Animaux , Technique de Western , Différenciation cellulaire , Division cellulaire , Lignée cellulaire tumorale , Clonage moléculaire , Exons , Analyse de profil d'expression de gènes , Humains , Leucémie aigüe myéloïde/anatomopathologie , Souris/génétique , Données de séquences moléculaires , Mutation , Séquençage par oligonucléotides en batterie , Retroviridae/génétique , RT-PCRRÉSUMÉ
The design and synthesis of glycol-functionalized porphyrins that contain one to four low molecular weight glycol chains that are linked via ether bonds to the meta-phenyl positions of meso-tetraphenylporphyrin and the comparison of fluorinated and nonfluorinated para derivatives are reported. The cellular uptake and photodynamic activity significantly depend on terminal groups of the glycol substituent. Hydroxy glycol porphyrins, in contrast with methoxy glycol porphyrins, show efficient intracellular transport and a high induction of apoptosis in tumor cell lines in vitro . Furthermore, the ethylene glycol chain at the meta position exhibits a superior efficacy that leads to the permanent ablation of human breast carcinoma (MDA-MB-231) in nude mice. In addition, fluorination enhanced the photosensitizing potential of para-phenyl derivatives. The analysis of the cell-death mechanism revealed that glycol-functionalized porphyrins represent novel nonmitochondrially localized photosensitizers that have a profound ability to induce apoptosis in tumor cells that act upstream of caspase activation. The strong interaction with a tumor marker (sialic acid) indicates the preferential association of these compounds with tumor cells.
Sujet(s)
Antinéoplasiques/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Glycols/pharmacologie , Tumeurs expérimentales/traitement médicamenteux , Photothérapie dynamique/méthodes , Porphyrines/pharmacologie , Animaux , Antinéoplasiques/administration et posologie , Antinéoplasiques/composition chimique , Lignée cellulaire tumorale , Conception de médicament , Tests de criblage d'agents antitumoraux , Cytométrie en flux , Glycols/administration et posologie , Glycols/composition chimique , Humains , Injections veineuses , Souris , Souris nude , Structure moléculaire , Masse moléculaire , Porphyrines/administration et posologie , Porphyrines/composition chimique , Stéréoisomérie , Facteurs temps , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS: Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.
Sujet(s)
Hématopoïèse/physiologie , Récepteurs à la thrombopoïétine/physiologie , Thrombopoïétine/physiologie , Séquence d'acides aminés , Animaux , Lignage cellulaire , Embryon de poulet , Poulets , Clonage moléculaire , Escherichia coli/génétique , Fibroblastes/cytologie , Humains , Souris , Données de séquences moléculaires , ARN messager/biosynthèse , Récepteurs à la thrombopoïétine/biosynthèse , Récepteurs à la thrombopoïétine/génétique , Protéines recombinantes/génétique , Alignement de séquences , Thrombopoïétine/biosynthèse , Thrombopoïétine/génétiqueRÉSUMÉ
Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.
Sujet(s)
Différenciation cellulaire , Phosphatidylsérine/métabolisme , Protéines proto-oncogènes c-myb/génétique , Animaux , Annexine A5/métabolisme , Séquence nucléotidique , Lignée cellulaire , Amorces ADN , Technique d'immunofluorescence , Souris , ARN messager/génétique , RT-PCRRÉSUMÉ
In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(beta-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(beta-CD)2. However, in vivo P(beta-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm(-2)). In contrast, P(beta-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(beta-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.
Sujet(s)
Antinéoplasiques/pharmacologie , Photosensibilisants/pharmacologie , Porphyrines/pharmacologie , Peau/effets des médicaments et des substances chimiques , Cyclodextrines bêta/pharmacologie , Animaux , Mort cellulaire/effets des médicaments et des substances chimiques , Mort cellulaire/effets des radiations , Dermatite phototoxique/traitement médicamenteux , Humains , Lysosomes/anatomopathologie , Souris , Souris de lignée BALB C , Peau/effets des radiations , Facteurs temps , Cellules cancéreuses en cultureRÉSUMÉ
Enhanced hydrolysis rates for the cleavage of bis(4-nitrophenyl)phosphate (BNPP), a model phosphodiester, may be achieved by using appropriately designed ditopic receptors containing the known phosphate-binding nucleus, sapphyrin, attached covalently to suitably oriented polyhydroxyl subunits. Evidence for the interaction between sapphyrin and BNPP comes from solid-state X-ray diffraction analysis of a diprotonated dihydroxylated sapphyrin-BNPP complex and from solution-phase (31)P NMR spectroscopic binding studies. The sapphyrins described in this paper may have a role to play as oligonucleotide cleavage agents.
Sujet(s)
Nitrophénols/composition chimique , Porphyrines/composition chimique , Catalyse , Liaison hydrogène , Hydrolyse , Hydroxylation , Cinétique , Structure moléculaire , Diffraction des rayons XRÉSUMÉ
Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.
Sujet(s)
Poulets , Tumeurs du rein/génétique , Oncogènes/génétique , Maladies de la volaille/génétique , Intégration virale/génétique , Tumeur de Wilms/génétique , Animaux , Virus de la myéloblastose aviaire/génétique , Protéines aviaires/génétique , Embryon de poulet , Cartographie chromosomique , Protéines de liaison à l'ADN/génétique , Gènes suppresseurs de tumeur , Gènes ras/génétique , Humains , Tumeurs du rein/virologie , Protéines oncogènes/génétique , Réaction de polymérisation en chaîne , Provirus/génétique , Séquences répétées terminales , Protéine-1 apparentée à Twist/génétique , Tumeur de Wilms/virologieRÉSUMÉ
c-Myb, known to play a central role in hematopoiesis, is also an important factor involved in myogenesis. Here, we found that the c-myb gene is expressed in proliferating C2C12 myoblasts and turned off in differentiating cells. Detailed analysis of c-myb RNAs revealed that the cell density is the essential factor determining c-myb expression. Both c-myb and its alternatively spliced form c-mybE9A RNAs are down-regulated in confluent cells. Constitutive expression of exogenous c-myb in C2C12 cells inhibits their terminal differentiation. It is shown that the c-Myb protein physically interacts with MyoD, the key regulator of myogenesis, and inhibits MyoD-dependent transcription. The interaction domains are the DNA binding domain of c-Myb and the bHLH motif of MyoD. Our data suggest that in proliferating cells c-Myb binds MyoD and inhibits its transcriptional activity until cell-cell contacts are established and c-myb expression is switched off. Thus, the c-Myb protein may be one of factors ensuring that proliferating myoblasts remain undifferentiated.
Sujet(s)
Différenciation cellulaire/physiologie , Protéine MyoD/antagonistes et inhibiteurs , Protéine MyoD/métabolisme , Myoblastes/métabolisme , Protéines proto-oncogènes c-myb/physiologie , Animaux , Numération cellulaire , Lignée cellulaire , Souris , Souris de lignée C3H , Myoblastes/cytologie , Facteurs de régulation myogènes/biosynthèse , Facteurs de régulation myogènes/génétique , Régions promotrices (génétique) , Liaison aux protéines , Protéines proto-oncogènes c-myb/biosynthèse , Protéines proto-oncogènes c-myb/génétiqueRÉSUMÉ
BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. AIM: The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. METHODS: As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. RESULTS: Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. CONCLUSIONS: Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.
Sujet(s)
Réarrangement des gènes des chaines lourdes des lymphocytes B/génétique , Chaines lourdes des immunoglobulines/génétique , Région variable d'immunoglobuline/génétique , Leucémie chronique lymphocytaire à cellules B/diagnostic , RT-PCR/méthodes , Benzothiazoles , Diamines , Femelle , Humains , Mâle , Maladie résiduelle , Composés chimiques organiques/analyse , Composés chimiques organiques/composition chimique , Quinoléines , ARN tumoral/analyse , ARN tumoral/composition chimique , Sensibilité et spécificitéRÉSUMÉ
Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.
Sujet(s)
Antigènes CD34/biosynthèse , Cytochrome P-450 enzyme system/génétique , Cellules souches hématopoïétiques/métabolisme , Cyclophiline A/biosynthèse , Cyclophiline A/génétique , Cytochrome P-450 enzyme system/biosynthèse , ADN complémentaire/isolement et purification , ADN complémentaire/métabolisme , Cytométrie en flux , Expression des gènes/génétique , Cellules souches hématopoïétiques/enzymologie , Cellules souches hématopoïétiques/immunologie , Humains , Immunotransfert , Isoenzymes/biosynthèse , Isoenzymes/génétique , ARN/isolement et purification , ARN/métabolisme , ARN messager/biosynthèse , ARN messager/génétiqueRÉSUMÉ
Novel cationic compounds forming complexes with oligodeoxyribonucleotides (ODNs) were prepared, and their ability to transport ODNs into cultured primary leukemic cells was tested. Two cationic porphyrin derivatives (2 and 3) were found to be at least 1 order of magnitude more efficient in this respect than commercially available agents. The ODN transporting capacity of novel compounds was dependent on the magnitude and the nature of their positive charges as well as on the porphyrin/ODN molar ratio. Porphyrin-ODN complexes were internalized into cells, and their dissociation was demonstrated by accumulation of fluorescein isothiocyanate-ODN fluorescence in the nucleus. Importantly, porphyrin 3 significantly protected complexed ODN against degradation and efficiently mediated the specific antisense effect on targeted v-Myb expression, resulting in reproducible growth inhibition of treated cells. Low toxicity, serum compatibility, and water solubility of porphyrin 3 make this compound a promising novel tool for modulation of gene expression in primary leukemic cells.
Sujet(s)
Oligodésoxyribonucléotides antisens/administration et posologie , Protéines oncogènes v-myb/génétique , Porphyrines/synthèse chimique , Animaux , Lignée de cellules transformées , Poulets , Vecteurs de médicaments , Fluorescéine-5-isothiocyanate , Techniques de transfert de gènes , Leucémies , Microscopie de fluorescence , Oligodésoxyribonucléotides antisens/composition chimique , Protéines oncogènes v-myb/métabolisme , Porphyrines/composition chimique , Relation structure-activité , Cellules cancéreuses en cultureRÉSUMÉ
GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.
Sujet(s)
Protéines de liaison à l'ADN/physiologie , Précurseurs érythroïdes/métabolisme , Érythropoïèse/physiologie , Protéines proto-oncogènes c-myb/physiologie , Protéines de répression/physiologie , Facteurs de transcription/physiologie , Animaux , Séquence nucléotidique , Sites de fixation , Protéines de transport/physiologie , Embryon de poulet , Poulets , Facteurs érythroïdes spécifiques , Fibroblastes , Facteur de transcription GATA-1 , Régulation de l'expression des gènes au cours du développement , Gènes myb , Humains , Cinétique , Données de séquences moléculaires , Protéines nucléaires/physiologie , Régions promotrices (génétique)/génétique , Liaison aux protéines , Proto-oncogène Mas , Protéines proto-oncogènes c-myb/génétique , Protéines de fusion recombinantes/physiologie , Facteur de croissance des cellules souches/pharmacologie , TransfectionRÉSUMÉ
The genes involved in the transformation of kidney blastema cells were searched for in avian nephroblastomas induced by the MAV2 retrovirus. The twist gene was identified as a common site of provirus integration in tumor cells. Twist was rearranged by the MAV2 provirus in three out of 76 independent nephroblastoma samples. The MAV2 integration sites were localized within 40 nucleotides of the twist 5'UTR region, right upstream from the ATG initiation codon. The integrated proviruses were deleted at their 5'ends. As a consequence, twist transcription became controlled by the retroviral 3'LTR promoter and was strongly upregulated, more than 200 times. In addition, 2-100 times elevated twist transcription was also detected in the majority of other nephroblastoma samples not containing MAV2 in the twist locus. We propose that chicken nephroblastoma originates from a single blastemic cell in which the MAV retrovirus, through its integration, has deregulated specific combinations of genes controlling proliferation and differentiation. The activation of the twist gene expression appears to contribute to tumorigenesis, as there is an in vivo positive selection of tumor cell clones containing the twist gene hyperactivated by MAV2 sequences inserted within the twist promoter.