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Front Oncol ; 8: 127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755954


Background and objective: Diffuse intrinsic pontine glioma (DIPG) is a lethal brainstem tumor in children. Dendritic cells (DCs) have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT) clinical trial with the use of autologous dendritic cells (ADCs) pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy). Methods: Nine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV) were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase. Results: Vaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients) and specific (8/9 patients) antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC). Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF) of two patients. Vaccine administration resulted safe in all patients treated with this schema. Conclusion: These preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

Cell Cycle ; 11(19): 3627-37, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22935704


Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38ß isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.

Replicação do DNA , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Mitose , Proteínas Quinases/metabolismo , Animais , Afidicolina/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Ciclina B1/antagonistas & inibidores , Ciclina B1/metabolismo , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Hidroxiureia/farmacologia , Cinética , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo
Cancer Res ; 67(24): 11648-56, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18089794


Cell cycle checkpoint abrogation, especially the inhibition of Chk1 in combination with DNA-damaging treatments, has been proposed as a promising way of sensitizing cancer cells. However, less is known about the possibility to selectively affect tumor cells when they are treated with agents that block DNA synthesis in combination with replication checkpoint inhibitors. Here, we present clear insights in the different responses of tumor and non-transformed cells to the inhibition of DNA replication with hydroxyurea in combination with checkpoint abrogation via inhibition of Ataxia telangiectasia-mutated- (ATM) and Rad3-related/ATM (ATR/ATM) and Chk1 kinases. Interestingly, we find that non-transformed cell lines activate ATR/ATM- and Chk1-independent pathways in response to replication inhibition to prevent mitotic entry with unreplicated DNA. In contrast, tumor cell lines such as HCT116 and HeLa cells rely entirely on Chk1 activity for a proper response to replication inhibitors. Our results show that p38 is activated in response to hydroxyurea treatment and collaborates with Chk1 to prevent mitotic entry in non-transformed cell lines by maintaining cyclin B1/Cdk1 complexes inactive. Furthermore, DNA replication arrest down-regulates cyclin B1 promoter activity in non-transformed cells, but not in tumor cells in a Chk1- and p38-independent way. Thus, our data show that non-transformed cells present a more robust DNA replication checkpoint response compared with tumor cells that involves activation of the p38 pathway. We show that some of these responses to replication block can be lost in tumor cells, causing a defective checkpoint and providing a rationale for tumor-selective effects of combined therapies.

Bromodesoxiuridina/farmacologia , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Fase S/efeitos dos fármacos , Células 3T3/citologia , Células 3T3/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Células HeLa , Humanos , Rim , Camundongos , RNA Interferente Pequeno/genética , Ratos
J Bacteriol ; 188(7): 2726-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16547063


Antibiotics that interfere with DNA replication and cell viability activate the SOS response. In Staphylococcus aureus, the antibiotic-induced SOS response promotes replication and high-frequency horizontal transfer of pathogenicity island-encoded virulence factors. Here we report that beta-lactams induce a bona fide SOS response in S. aureus, characterized by the activation of the RecA and LexA proteins, the two master regulators of the SOS response. Moreover, we show that beta-lactams are capable of triggering staphylococcal prophage induction in S. aureus lysogens. Consequently, and as previously described for SOS induction by commonly used fluoroquinolone antibiotics, beta-lactam-mediated phage induction also resulted in replication and high-frequency transfer of the staphylococcal pathogenicity islands, showing that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors.

Antibacterianos/farmacologia , Transferência Genética Horizontal/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/metabolismo , beta-Lactamas/farmacologia , Ampicilina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Penicilinas/farmacologia , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/genética
J Bacteriol ; 187(15): 5367-75, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16030231


The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the delta-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other delta-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the delta-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.

Proteínas de Bactérias/genética , Bdellovibrio/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Ligação Proteica , Proteínas Repressoras/metabolismo , Serina Endopeptidases/metabolismo
Nucleic Acids Res ; 32(22): 6617-26, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15604457


The LexA regulon encompasses an ensemble of genes involved in preserving cell viability under massive DNA damage and is present in most bacterial phyla. Up to date, however, the scope of this network had only been assessed in the Gamma Proteobacteria. Here, we report the structure of the LexA regulon in the Alpha Proteobacteria, using a combined approach that makes use of in vitro and in vivo techniques to assist and validate the comparative genomics in silico methodology. This leads to the first experimentally validated description of the LexA regulon in the Alpha Proteobacteria, and comparison of regulon core structures in both classes suggests that a least common multiple set of genes (recA, ssb, uvrA and ruvCAB) might be a defining property of the Proteobacteria LexA network.

Alphaproteobacteria/genética , Proteínas de Bactérias/metabolismo , Gammaproteobacteria/genética , Genômica/métodos , Regulon , Serina Endopeptidases/metabolismo , Algoritmos , Sequência de Bases , Sítios de Ligação , Biologia Computacional , Sequência Consenso , Sequências Reguladoras de Ácido Nucleico