RESUMO
DAP kinase is a pro-apoptotic calcium-regulated serine/threonine kinase, whose expression is frequently lost in human tumours. Here we show that DAP kinase counteracts oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint. Ectopic expression of DAP kinase suppressed oncogenic transformation of primary embryonic fibroblasts by activating p53 in a p19ARF-dependent manner. Consequently, the fibroblasts underwent apoptosis, characterized by caspase activation and DNA fragmentation. In response to c-Myc or E2F-1, the endogenous DAP kinase protein was upregulated. Furthermore, functional or genetic inactivation of the endogenous DAP kinase reduced the extent of induction of p19ARF/p53 and weakened the subsequent apoptotic responses to c-Myc or E2F-1. These results establish a role for DAP kinase in an early apoptotic checkpoint designed to eliminate pre-malignant cells during cancer development.
Assuntos
Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/deficiência , Proteínas de Transporte , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA , Genes Supressores de Tumor/fisiologia , Genes cdc/fisiologia , Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Divisão Celular/genética , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/enzimologia , Transformação Celular Neoplásica/genética , Proteínas Quinases Associadas com Morte Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Feto , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes myc/fisiologia , Camundongos , Camundongos Knockout , Oncogenes/fisiologia , Proteínas/genética , Proteína 1 de Ligação ao Retinoblastoma , Transdução de Sinais/genética , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/genéticaRESUMO
Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/fisiologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Inibidores de Caspase , Caspases/genética , Caspases/metabolismo , Linhagem Celular , Proteínas Quinases Associadas com Morte Celular , Proteína de Domínio de Morte Associada a Fas , Genes Dominantes/genética , Humanos , Proteínas Inibidoras de Apoptose , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , RNA Antissenso/genética , RNA Antissenso/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Serpinas/genética , Serpinas/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteínas Virais/genética , Proteínas Virais/fisiologia , Receptor fas/genéticaRESUMO
This article describes the case of a patient who suffered an electrical full thickness burn of the chest wall and a concomitant osteomyelitic complication of two ribs. A review of the existing literature on bone and joint changes after burns is presented. Osteomyelitis of ribs must be kept in mind while treating patients for chest wall burns.
Assuntos
Queimaduras por Corrente Elétrica/complicações , Osteomielite/etiologia , Adulto , Queimaduras por Corrente Elétrica/cirurgia , Humanos , Masculino , Osteomielite/cirurgia , CostelasRESUMO
A simple method of umbilical repositioning by incising the anterior rectus sheath and rectus abdominis muscle is reported for cases of unilateral abdominal wall plication during the TRAM flap operation. This method keeps the umbilicus stable and nonstenotic, and it avoids hypertrophic scars, which result from other techniques such as direct suturing of the stalk to the skin. Although this method might weaken contralateral muscle activity, the patients we operated on maintained their ability to perform sit-ups, and no periumbilical weakening was noticed.
Assuntos
Músculos Abdominais/cirurgia , Retalhos Cirúrgicos , Umbigo/cirurgia , HumanosRESUMO
Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspases/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases Associadas com Morte Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Camundongos , Camundongos Knockout , Fosfosserina/metabolismo , Tunicamicina/administração & dosagem , Tunicamicina/toxicidadeRESUMO
New pigmented lesions with alarming properties should trigger the responsible physician to perform diagnostic procedures including biopsies, blood tests, and endocrinological evaluations. A unique pigmented dermatosis of unknown etiology, known as terra firma forme dermatosis, creates a cosmetic disturbance that might mislead experienced physicians and trigger unnecessary and expensive workups when all that is needed is a firm rubbing with 70% alcohol-impregnated applicators. We present a 17-year-old girl with such a lesion and discuss the diagnostic possibilities.
Assuntos
Dermatomicoses/diagnóstico , Malassezia , Transtornos da Pigmentação/diagnóstico , Adolescente , Dermatomicoses/terapia , Diagnóstico Diferencial , Feminino , Humanos , Transtornos da Pigmentação/terapiaRESUMO
A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RNAs, was employed to identify mediators of interferon-gamma-induced apoptosis. One of the several genes identified is DAP3, which codes for a 46 kDa protein with a potential nucleotide-binding motif. Structure-function studies of the protein indicate that the intact full-length protein is required for its ability to induce apoptosis when overexpressed. The N-terminal 230 amino acids, on the other hand, act in a dominant-negative fashion. Both of these functions are dependent on the integrity of the nucleotide binding motif. Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas and tumor necrosis factor alpha (TNF-alpha) receptors. Thus, DAP3 is implicated as a positive mediator of these death-inducing stimuli. It functions downstream of the receptor signaling complex and its death promoting effects depend on caspase activity. In the nematode Caenorhabditis elegans, a potential homolog of DAP3 showing 35% identity and 64% similarity to the human protein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian cells induced cell death, indicating that the protein is conserved at the functional level as well as the structural level.
Assuntos
Apoptose/fisiologia , Proteínas/química , Proteínas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Caspases/fisiologia , Evolução Molecular , Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteínas/genética , RNA Antissenso/genética , RNA Antissenso/farmacologia , Proteínas de Ligação a RNA , Proteínas Ribossômicas , Homologia de Sequência , Transdução de Sinais , Especificidade da Espécie , TransfecçãoRESUMO
The antiproliferative functions of interferons result from specific effects that these cytokines exert on several cell cycle-controlling genes. The possible coupling between the interferon-responsive genes that are directly transactivated by the interferon signaling and the genes that constitute the basic machinery of the cell cycle is not clear yet. We report in this work that interferon-induced double-stranded RNA-activated kinase (PKR) is one of the specific mediators of the antiproliferative effects of the cytokine. Transfections of M1 myeloid leukemia cells with two catalytically inactive mutant forms of PKR abrogated the ability of interferon to suppress c-Myc without interfering with the pRB/cyclin D responses. As a consequence, these genetically manipulated cells displayed a small but significant reduction in their growth sensitivity to interferons, a phenotype that characterizes a single pathway disruption. Transfection of the parental M1 cells with the functional wild-type human PKR restricted their proliferation in the absence of interferons. This PKR-mediated growth inhibition could be efficiently rescued by the ectopic expression of deregulated c-myc. Taken together these results prove the existence of direct or indirect links between PKR and c-Myc suppression, thereby placing this gene along one of the complementary growth suppressive pathways that are triggered by interferons.
Assuntos
Interferon-alfa/farmacologia , Interferon beta/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Animais , Linfoma de Burkitt , Linhagem Celular , Indução Enzimática/efeitos dos fármacos , Humanos , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Supressão Genética , Transfecção , Células Tumorais Cultivadas , eIF-2 QuinaseRESUMO
Death-associated protein kinase (DAP-kinase) is a Ca(+2)/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-gamma to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-alpha-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the "linker" region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.
Assuntos
Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Associadas com Morte Celular , Testes Genéticos , Humanos , Interferon gama/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Receptor de Fator de Crescimento Neural/química , Alinhamento de Sequência , Deleção de Sequência , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.
Assuntos
Morte Celular/fisiologia , Expressão Gênica , Interferon gama/farmacologia , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos , Proteínas Reguladoras de Apoptose , Sequência de Bases , Morte Celular/efeitos dos fármacos , Divisão Celular , Clonagem Molecular , DNA Antissenso , DNA Complementar , Biblioteca Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas/genética , Proteínas/isolamento & purificação , RNA Antissenso/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA , Coelhos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Reticulócitos/metabolismo , Proteínas Ribossômicas , TransfecçãoRESUMO
To assess the efficacy of the topical anesthetic cream, EMLA, in alleviating the pain produced by infiltration of local anesthetic prior to surgical skin biopsies, a randomized, double-blind, placebo-controlled study was performed on 54 patients undergoing 162 excisional biopsies. Both pain induced by needle insertion and pain induced by local injection were significantly diminished after topical application of EMLA cream. However, part of the effect was placebo, because the placebo ointment (Vaseline) also produced significant pain alleviation.
Assuntos
Anestesia Local , Anestésicos Locais , Biópsia , Lidocaína , Medição da Dor , Prilocaína , Pele/patologia , Adulto , Combinação de Medicamentos , Feminino , Humanos , Injeções , Combinação Lidocaína e Prilocaína , Masculino , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
Forty-three women, in a series of 150, participated in a prospective study that examined their chest walls for deformities 3 months after maximal tissue expansion for single-breast reconstruction. Computed tomography imaging was used for this purpose. Twenty-one patients underwent immediate breast reconstruction and the other 22 patients underwent delayed reconstruction. Fifty-three percent had some chest wall abnormality. In the delayed group, chest wall deformities were more statistically significant (p < 0.001). Our findings suggest that chest wall deformity is a common occurrence after maximal tissue expansion for breast reconstruction.
Assuntos
Tórax em Funil/diagnóstico por imagem , Mamoplastia/instrumentação , Complicações Pós-Operatórias/diagnóstico por imagem , Dispositivos para Expansão de Tecidos , Tomografia Computadorizada por Raios X , Adulto , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/cirurgia , Feminino , Seguimentos , Humanos , Mastectomia , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
This article describes a rare malignant schwannoma invading the left mandible in a 42-year-old man. The clinical presentation, inconclusive radiographic findings, and light microscopic histology are included. The appropriate surgical treatment could be determined only intraoperatively when frozen sections provided the exact tumor margins. The patient subsequently received adjuvant treatment by brachytherapy.