RESUMO
Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking, and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings. Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.
Assuntos
Proteínas Hedgehog , Neoplasias Ovarianas , Animais , Feminino , Humanos , Camundongos , Proteínas de Ligação ao Cálcio/metabolismo , Transformação Celular Neoplásica , Proteínas da Matriz Extracelular/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Recidiva Local de Neoplasia , Neoplasias Ovarianas/metabolismo , Microambiente Tumoral , Proteína de Matriz GlaRESUMO
Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Receptores ErbB/química , Fosfopeptídeos/química , Proteínas/química , Proteínas Proto-Oncogênicas c-met/química , Domínios de Homologia de src , Sequência de Aminoácidos , Sequência Conservada , Cristalização , Cristalografia por Raios X , Dimerização , Proteína Adaptadora GRB2 , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Triptofano/química , Água/químicaRESUMO
A set of O-substituted aryl beta-D-glucopyranosides were prepared and found to have inhibitory activity on the growth of two carcinoma cell lines.
Assuntos
Antineoplásicos/síntese química , Glucosídeos/síntese química , Domínios de Homologia de src/efeitos dos fármacos , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Glucosídeos/farmacologia , Células HT29 , Humanos , Ligantes , Conformação Molecular , Células Tumorais CultivadasRESUMO
Hepatocyte growth factor (HGF) induces a three-phase response leading to the formation of branched tubular structures in epithelial cells. The HGF receptor tyrosine kinase works through a Src homology (SH2) docking site that can activate several signalling pathways. The first phase of the response (scattering), which results from cytoskeletal reorganization, loss of intercellular junctions and cell migration, is dependent on phosphatidylinositol-3-OH kinase and Rac activation. The second phase (growth) requires stimulation of the Ras-MAP kinase cascade. Here we show that the third phase (tubulogenesis) is dependent on the STAT pathway. HGF stimulates recruitment of Stat-3 to the receptor, tyrosine phosphorylation, nuclear translocation and binding to the specific promoter element SIE. Electroporation of a tyrosine-phosphorylated peptide, which interferes with both the association of STAT to the receptor and STAT dimerization, inhibits tubule formation in vitro without affecting either HGF-induced 'scattering' or growth. The same result is obtained using a specific 'decoy' oligonucleotide that prevents STAT from binding to DNA and affecting the expression of genes involved in cell-cycle regulation (c-fos and waf-1). Activation of signal transducers that directly control transcription is therefore required for morphogenesis.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Células Epiteliais/citologia , Fator de Crescimento de Hepatócito/fisiologia , Transativadores/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Eletroporação , Regulação da Expressão Gênica , Morfogênese/fisiologia , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/antagonistas & inibidores , Transativadores/metabolismoRESUMO
RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.
Assuntos
Proteínas de Drosophila , Isoenzimas/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Fosfolipases Tipo C/metabolismo , Tirosina , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Glutationa Transferase/biossíntese , Células HeLa , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Oncogênicas/biossíntese , Fenilalanina , Fosfopeptídeos/química , Fosforilação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/biossíntese , TransfecçãoRESUMO
The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.
Assuntos
Antibióticos Antineoplásicos/farmacocinética , DNA/efeitos dos fármacos , Oligonucleotídeos/metabolismo , Sequência de Bases , Sítios de Ligação , Fenômenos Químicos , Química , Daunorrubicina/farmacocinética , Doxorrubicina/farmacocinética , Espectrometria de FluorescênciaRESUMO
During cholecystectomy 21 patients were subjected to transcystic extraction of common bile duct stones by a Dormia-Pironneau catheter; this was the only treatment used for lithiasis of the common bile duct, in the absence of papillary lesions. Of these patients, 14 were followed up from one to three years after surgery. Clinical and radiological controls revealed the patients' complete recovery from the biliary disease. Our late results thus warrant the method. We suggest that is should be employed in all cases in which the presence of minute stones in the gallbladder, a wide cystic duct, and the absence of papillary lesions suggest migration of gallstones from the gallbladder to the common bile duct.
Assuntos
Cateterismo/métodos , Cálculos Biliares/cirurgia , Ducto Cístico/cirurgia , Humanos , Cuidados IntraoperatóriosAssuntos
Doenças Biliares/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Fosfatidilcolinas/uso terapêutico , Colelitíase/tratamento farmacológico , Colestase/tratamento farmacológico , Combinação de Medicamentos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Metástase Neoplásica , Pancreatite/tratamento farmacológico , Complexo Vitamínico B/uso terapêuticoRESUMO
With a view to carrying out a critical review of the use of instrumental divulsion in surgery of the bile ducts, a long-term follow-up was made, from one to 12 years after performance of this operation, in 103 out of 157 cases. An examination of the immediate results which were favourable, in contrast with those found at long-term, led to the conclusion that this procedure was of little use and it has now been dropped both in diagnostics and, with greater reson, in treatment of benign papillary stenosis.