RESUMEN
BACKGROUND: In the phase III KEYNOTE-189 study (NCT02578680), pembrolizumab plus pemetrexed and platinum-based chemotherapy (pemetrexed-platinum) significantly improved overall survival (OS) and progression-free survival (PFS) in patients with previously untreated metastatic nonsquamous non-small-cell lung cancer (NSCLC) versus placebo plus pemetrexed-platinum. We report updated efficacy outcomes from the protocol-specified final analysis, including outcomes in patients who crossed over to pembrolizumab from pemetrexed-platinum and in patients who completed 35 cycles (â¼2 years) of pembrolizumab. PATIENTS AND METHODS: Eligible patients were randomized 2 : 1 to receive pembrolizumab 200 mg (n = 410) or placebo (n = 206) every 3 weeks (for up to 35 cycles, â¼2 years) plus four cycles of pemetrexed (500 mg/m2) and investigators' choice of cisplatin (75 mg/m2) or carboplatin (area under the curve 5 mg·min/ml) every 3 weeks, followed by pemetrexed until progression. Patients assigned to placebo plus pemetrexed-platinum could cross over to pembrolizumab upon progression if eligibility criteria were met. The primary endpoints were OS and PFS. RESULTS: After a median follow-up of 31.0 months, pembrolizumab plus pemetrexed-platinum continued to improve OS [hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.46-0.69] and PFS (HR, 0.49; 95% CI, 0.41-0.59) over placebo plus pemetrexed-platinum regardless of programmed death-ligand 1 expression. Objective response rate (ORR) (48.3% versus 19.9%) and time to second/subsequent tumor progression on next-line treatment (PFS2; HR, 0.50; 95% CI, 0.41-0.61) were improved in patients who received pembrolizumab plus pemetrexed-platinum. Eighty-four patients (40.8%) from the placebo plus pemetrexed-platinum group crossed over to pembrolizumab on-study. Grade 3-5 adverse events occurred in 72.1% of patients receiving pembrolizumab plus pemetrexed-platinum and 66.8% of patients receiving placebo plus pemetrexed-platinum. Fifty-six patients completed 35 cycles (â¼2 years) of pembrolizumab; ORR was 85.7% and 53 (94.6%) were alive at data cut-off. CONCLUSIONS: Pembrolizumab plus pemetrexed-platinum continued to show improved efficacy outcomes compared with placebo plus pemetrexed-platinum, with manageable toxicity. These findings support first-line pembrolizumab plus pemetrexed-platinum in patients with previously untreated metastatic nonsquamous NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Pemetrexed/uso terapéutico , Platino (Metal)/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Background: Analysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib. Patients and methods: Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS. Results: Thirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for ≥24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR. Conclusions: Monitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , ADN Tumoral Circulante/genética , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Quinazolinas/uso terapéutico , Adenocarcinoma/sangre , Adenocarcinoma/enzimología , Adenocarcinoma del Pulmón , Afatinib , ADN Tumoral Circulante/sangre , Receptores ErbB/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/enzimología , Masculino , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa/métodos , Estudios Prospectivos , Quinazolinas/efectos adversosRESUMEN
We describe successful treatment of 3 cases of human herpesvirus 6 (HHV-6) encephalitis/myelitis following cord blood transplantation (CBT). Ganciclovir (GCV) (10 mg/kg/day) reduced HHV-6 load to undetectable levels in cerebrospinal fluid (CSF). Early dose reduction in the presence of HHV-6 detectable in CSF resulted in an increased HHV-6 load. GCV was capably shifted to valganciclovir (VGCV) with an almost equivalent concentration. GCV/VGCV may be effective for HHV-6 encephalitis/myelitis after CBT, although HHV-6 load in CSF should be monitored.
Asunto(s)
Antivirales/uso terapéutico , Encefalitis Viral/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 6/aislamiento & purificación , Mielitis/tratamiento farmacológico , Infecciones por Roseolovirus/tratamiento farmacológico , Acondicionamiento Pretrasplante/efectos adversos , Carga Viral/efectos de los fármacos , Adulto , Antivirales/administración & dosificación , Preescolar , ADN Viral , Encefalitis Viral/líquido cefalorraquídeo , Encefalitis Viral/virología , Femenino , Sangre Fetal , Ganciclovir/administración & dosificación , Ganciclovir/análogos & derivados , Ganciclovir/uso terapéutico , Humanos , Masculino , Mielitis/líquido cefalorraquídeo , Mielitis/virología , Agonistas Mieloablativos/efectos adversos , Infecciones por Roseolovirus/líquido cefalorraquídeo , Infecciones por Roseolovirus/virología , Resultado del Tratamiento , Valganciclovir , Adulto JovenRESUMEN
BACKGROUND: We evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, recommended dose for phase II (P2RD), and preliminary anticancer activity of a combination eribulin and S-1 therapeutic in metastatic breast cancer patients pretreated with anthracycline and taxane. METHOD: Patients aged 20-74 years were recruited. In level 1, patients received S-1 (65 mg m(-2)) from day 1 to 14, and eribulin (1.1 mg m(-2)) on day 1 and 8 in a 21-day cycle. In level 2, eribulin was increased to 1.4 mg m(-2). In level 3, S-1 was increased to 80 mg m(-2). RESULTS: Twelve patients were enrolled into three cohorts. Planned dose escalation was completed, with one case exhibiting dose-limiting toxicity (grade 3 hypokalaemia) at level 3, without reaching the MTD. The P2RD was determined to be level 2 (eribulin 1.4 mg m(-2) and S-1 65 mg m(-2)). The most common grade 3 or 4 toxicity was neutropenia (83.3%), followed by febrile neutropenia (25.0%). Five of eleven patients (41.7%) with measurable disease had a partial response. Pharmacokinetics were characterised by dose-dependent elimination and nonlinear exposure. CONCLUSION: Dose level 3 was not tolerated owing to febrile neutropenia development. Thus, intermediate dose level 2 was recommended for further evaluation. Preliminary antitumour activity warrants further investigation in this setting.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Furanos/administración & dosificación , Furanos/farmacocinética , Cetonas/administración & dosificación , Cetonas/farmacocinética , Ácido Oxónico/administración & dosificación , Ácido Oxónico/farmacocinética , Tegafur/administración & dosificación , Tegafur/farmacocinética , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Combinación de Medicamentos , Femenino , Furanos/uso terapéutico , Humanos , Cetonas/uso terapéutico , Dosis Máxima Tolerada , Persona de Mediana Edad , Metástasis de la Neoplasia/tratamiento farmacológico , Ácido Oxónico/uso terapéutico , Tegafur/uso terapéutico , Adulto JovenRESUMEN
BACKGROUND: A variety of hereditary spinocerebellar ataxia (SCA) develops a broad spectrum of both ataxia and non-ataxia symptoms. Cognitive and affective changes are one such non-ataxia symptoms, but have been described only in hereditary SCAs with exonic CAG gene expansion. METHODS: We newly found intronic hexanucleotide GGCCTG gene expansion in NOP56 gene as the causative mutation (=SCA36) in nine unrelated Japanese familial SCA originating from Asida river area in the western part of Japan, thus nicknamed Asidan for this mutation. These patients show unique clinical balance of cerebellar ataxia and motor neuron disease (MND), locating on the crossroad of these two diseases. In the nine families, 14 patients were clinically examined and genetically confirmed to Asidan. In the present study, we examined cognitive and affective analyses on 12 patients (seven men and five women) who agreed to join the examination with average age at onset of 53.1 ± 3.2 years, average duration of 12.1 ± 5.2 years, and current average age at 65.1 ± 6.2 years. RESULTS: The 12 Asidan patients demonstrated a significant decrease in their frontal executive functions measured by frontal assessment battery (FAB) and Montreal cognitive assessment (MoCA) compared with age- and gender-matched controls, whilst mini-mental state examination (MMSE) and Hasegawa dementia score-revised (HDS-R) were within normal range. The decline of frontal executive function was related to their disease duration and scale for the assessment and rating of ataxias (SARA). They also demonstrated mild depression and apathy. Single-photon emission tomography (SPECT) analysis showed that these Asidan patients showed decline of regional cerebral blood flow (rCBF) in a particular areas of cerebral cortices such as Brodmann areas 24 and 44-46. CONCLUSION: These data suggest that the patients with Asidan mutation show unique cognitive and affective characteristics different from other hereditary SCAs with exonal CAG expansion or MND.
Asunto(s)
Trastornos del Conocimiento/genética , Enfermedad de la Neurona Motora/complicaciones , Enfermedad de la Neurona Motora/genética , Mutación , Proteínas Nucleares/genética , Ataxias Espinocerebelosas/complicaciones , Ataxias Espinocerebelosas/genética , Edad de Inicio , Anciano , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Mucosal immunoglobulin (Ig)A dominance has been proposed to be associated with preferential class switch recombination (CSR) to the IgA heavy chain constant region, Calpha. Here, we report that B cell activation in nasal-associated lymphoid tissue (NALT) upon stimulation with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma globulin caused an anti-NP memory response dominated by high affinity IgA antibodies. In the response, however, NP-specific IgG(+) B cells expanded and sustained their number as a major population in germinal centers (GCs), supporting the view that CSR to IgG heavy chain constant region, Cgamma, operated efficiently in NALT. Both IgG(+) and IgA(+) GC B cells accumulated somatic mutations, indicative of affinity maturation to a similar extent, suggesting that both types of cell were equally selected by antigen. Despite the selection in GCs, high affinity NP-specific B cells were barely detected in the IgG memory compartment, whereas such cells dominated the IgA memory compartment. Taken together with the analysis of the V(H) gene clonotype in GC and memory B cells, we propose that NALT is equipped with a unique machinery providing IgA-specific enrichment of high affinity cells into the memory compartment, facilitating immunity with high affinity and noninflammatory secretory antibodies.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Inmunoglobulina A/inmunología , Memoria Inmunológica/inmunología , Cavidad Nasal/inmunología , Administración Intranasal , Animales , Antígenos/inmunología , Antígenos/farmacología , Células Cultivadas , Quimiotaxis de Leucocito , Centro Germinal/inmunología , Haptenos/inmunología , Haptenos/farmacología , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas , Inyecciones Intraperitoneales , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Nasal/citología , Nitrofenoles/inmunología , Nitrofenoles/farmacología , FenilacetatosRESUMEN
BACKGROUND: It remains unclear whether response rate (RR) is related to survival benefit in phase III trials of advanced cancer treated with molecular targeted agents (MTA) in combination with standard therapies. MATERIALS AND METHODS: We carried out a systematic search of PubMed for randomized phase III trials of four solid tumors examining the efficacy of MTA when added to a standard therapy. We examined whether there were any associations between RR increment obtained by the addition of targeted agents (DeltaRR) and survival benefit in phase III trials. RESULTS: We identified 26 phase III trials of MTA with a total of 21 156 patients and 29 experimental arms of MTA. Studies which showed significant survival benefit had higher DeltaRR compared with those which did not show significant benefit. In the receiver operating characteristic curve analysis, using a 7% gain as threshold value for DeltaRR allowed assessment of survival benefit with high sensitivity and specificity. There were also significant relationships between DeltaRR and hazard ratios for overall survival and progression-free survival in the linear regression analysis. CONCLUSION: RR increment obtained by the addition of MTA to a standard therapy may be useful to predict survival benefit in clinical phase III trials of advanced cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Ensayos Clínicos Fase III como Asunto , Neoplasias/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Humanos , Análisis de SupervivenciaRESUMEN
AIM: Minor recurrent aphthous stomatitis (MRAS) is a common, painful and inflammatory ailment of the oral cavity with juvenile onset and unknown aetiology. The purpose of this study was to evaluate the potential of ascorbate (vitamin C) to reduce the frequency of MRAS and severity of pain. PATIENTS AND METHODS: Sixteen MRAS patients (9 boys and 7 girls: mean age, 12.0 +/- 2.4 years old) were assigned to take an oral dosage of 2000 mg/m(2)/day ascorbate. SUBJECTS: Their baseline frequency of outbreaks and the level of pains were compared during the treatment; in addition, a crossover clinical trial was performed. Polymorphonuclear leucocytes play a role in the pathogenesis, and then superoxide anion production was evaluated in prior to ascorbate treatment. RESULTS: The data indicated a statistically significant 50% reduction in oral ulcer outbreaks and a decline of pain level. Neutrophils were primed for superoxide anion production in the patients with MRAS. CONCLUSION: Ascorbate may modulate the generation of reactive oxygen species and augment neutrophil apoptosis, which could prevent neutrophil-mediated inflammation. Ascorbate seems to be effective, but the findings of our study were preliminary and it should be re-evaluated with a larger randomized controlled clinical trials.
Asunto(s)
Antioxidantes/uso terapéutico , Ácido Ascórbico/uso terapéutico , Estomatitis Aftosa/prevención & control , Administración Oral , Adolescente , Niño , Estudios Cruzados , Esquema de Medicación , Femenino , Humanos , Masculino , Neutrófilos/metabolismo , Dolor/prevención & control , Prevención Secundaria , Índice de Severidad de la Enfermedad , Superóxidos/metabolismo , Resultado del TratamientoAsunto(s)
Enfermedades del Sistema Nervioso Autónomo/genética , Trastornos del Conocimiento/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades por Prión/genética , Adulto , Enfermedades del Sistema Nervioso Autónomo/complicaciones , Trastornos del Conocimiento/complicaciones , Femenino , Eliminación de Gen , Humanos , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades por Prión/complicacionesRESUMEN
Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. We previously found that the transcript level of the nitrite reductase gene of F. oxysporum, named FoNIIA, was markedly upregulated during conidiation compared with during vegetative growth. FoNIIA was also found to be positively regulated by Ren1 that is a transcription regulator controlling development of microconidia and macroconidia. In this study, we analyzed the function of FoNIIA in conidiation of F. oxysporum. Conidiation cultures showed markedly higher level of accumulation of FoNiiA protein as well as FoNIIA mRNA than vegetative growth cultures. FoNIIA protein was significantly decreased in cultures of the REN1 disruption mutant compared with that of the wild type. These results confirmed that FoNIIA expression is upregulated during conidiation and is positively regulated by REN1. The FoNIIA disruption mutants produced microconidia, macroconidia, and chlamydospores, which were morphologically indistinguishable from those of the wild type. The mutants, however, produced significantly fewer macroconidia than the wild type, although the wild type and mutant strains produced similar numbers of microconidia and chlamydospores. These results demonstrate that nitrite reductase is involved in quantitative control of macroconidium formation as well as nitrate utilization in F. oxysporum.
Asunto(s)
Fusarium/genética , Nitrito Reductasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Cartilla de ADN , Oscuridad , Proteínas Fúngicas/genética , Fusarium/enzimología , Fusarium/crecimiento & desarrollo , Luz , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microbiología del Suelo , Solanum tuberosum/microbiología , Esporas Fúngicas/enzimología , Esporas Fúngicas/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.
Asunto(s)
ADN/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Oncogénicas de Retroviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN/aislamiento & purificación , Embrión de Mamíferos , Femenino , Biblioteca de Genes , Humanos , Pulmón , Datos de Secuencia Molecular , Neoplasias , Oligodesoxirribonucleótidos , Proteína Oncogénica v-crk , Placenta/fisiología , Plásmidos , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas Proto-Oncogénicas c-crk , Empalme del ARN , ARN Mensajero/genética , TransfecciónRESUMEN
Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.
Asunto(s)
Neuronas/citología , Proteínas Oncogénicas de Retroviridae/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Neuritas/metabolismo , Proteína Oncogénica v-crk , Células PC12 , Proteínas Oncogénicas de Retroviridae/químicaRESUMEN
It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.
Asunto(s)
Proteínas Quinasas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido , Humanos , Técnicas In Vitro , Factores de Crecimiento Nervioso/farmacología , Células PC12 , Unión Proteica , Conejos , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.
Asunto(s)
Membrana Celular/efectos de los fármacos , Proteínas/farmacología , Proteínas Oncogénicas de Retroviridae/metabolismo , Proteínas de Unión al GTP rac , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , Clonación Molecular , Receptores ErbB/metabolismo , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteína Oncogénica v-crk , Biosíntesis de Proteínas , Proteínas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Oncogénicas de Retroviridae/genéticaRESUMEN
Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.
Asunto(s)
Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Células 3T3 , Animales , Activación Enzimática , Ratones , Mutación , Ratas , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap1/genéticaRESUMEN
A profile of the the National Institute of Infectious Diseases (NIID) at Toyama Shinjuku-Ku, Tokyo, Japan.
Asunto(s)
Academias e Institutos/organización & administración , Enfermedades Transmisibles , Agencias Gubernamentales/organización & administración , Investigación/organización & administración , Bacteriología , Cooperación Internacional , Japón , Parasitología , Virología , Organización Mundial de la SaludRESUMEN
Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180; however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , ADN Complementario/química , Proteínas Activadoras de GTPasa , Humanos , Inmunohistoquímica , Leucocitos/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas Quinasas/metabolismo , Proteínas/química , Proteínas Proto-Oncogénicas c-crk , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Bazo/metabolismo , Timo/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
PURPOSE: RPR 109881A is a new semisynthetic taxoid compound that has a similar mechanism of action to docetaxel. The purpose of this phase I study was to characterize the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetic profile, and antitumor effects of this agent. PATIENTS AND METHODS: Nineteen eligible patients with advanced solid tumors were enrolled. RPR 109881A was administered as a 1-hour intravenous infusion every 3 weeks at doses ranging from 15 to 75 mg/m(2). Pharmacokinetic evaluation was performed at the first cycle. RESULTS: Neutropenia (febrile neutropenia) and fatigue were dose-limiting toxicities at doses of 60 and 75 mg/m(2) and seemed to be dose-related. Both thrombocytopenia and anemia were infrequent. Nonhematologic toxicities were generally mild. Pharmacokinetic studies indicated that RPR 109881A plasma disposition was bi- or triphasic, with a high total plasma clearance, a large volume of distribution, and a long terminal half-life. The area under the concentration-time curve (AUC) and the peak concentration of RPR 109881A seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics. Urinary excretion over 48 hours was low, with a mean of 0.8 +/- 0.36% of the administered dose. A significant relationship existed between the percentage decrease of neutrophil counts and the AUC of RPR 109881A. Among 18 assessable patients, two partial and two minor responses were documented. CONCLUSION: RPR 109881A was found to be a well-tolerated and promising taxoid agent. The MTD was 75 mg/m(2), and the recommended dose for phase II study was 60 mg/m(2) as a 1-hour infusion every 3 weeks.
Asunto(s)
Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/farmacocinética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Paclitaxel/efectos adversos , Paclitaxel/farmacocinética , Taxoides , Adulto , Anciano , Antineoplásicos Fitogénicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Paclitaxel/análogos & derivadosRESUMEN
v-Crk, identified as an oncogene product of the CT10 retrovirus, became the first example of an adaptor protein. It consists mostly of the Src homology 2 (SH2) and Src homology 3 (SH3) domains. Two of the three major proteins bound to Crk SH2 have been identified as paxillin and p130Cas. Both paxillin and p130Cas are phosphorylated upon stimulation by integrin, suggesting that Crk transduces signals from integrin. The cloning of the complementary DNA of two major proteins bound to Crk SH3 was recently completed. Both cDNAs encoded novel proteins: C3G, a guanine nucleotide exchange protein for Rap1, and DOCK180, an SH3-containing protein of unknown function. The SH3 domain of Crk also binds to Sos, Abl, and Eps15. The variety of the proteins bound to Crk SH3 implies that Crk provides a set of effector proteins that are triggered together. Alternatively, other domains of the SH3-binding proteins enable Crk to specifically activate each of the SH3-binding proteins according to the particular form of stimulation.
Asunto(s)
Proteínas Oncogénicas de Retroviridae/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Proteína Oncogénica v-crk , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Transformación GenéticaRESUMEN
Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.