RESUMEN
BACKGROUND: We conducted a phase I, multicenter, open-label, dose-finding, and expansion study to determine the safety and preliminary efficacy of eprenetapopt (APR-246) combined with pembrolizumab in patients with advanced/metastatic solid tumors (ClinicalTrials.gov NCT04383938). PATIENTS AND METHODS: For dose-finding, requirements were non-central nervous system primary solid tumor, intolerant to/progressed after ≥1 line of treatment, and eligible for pembrolizumab; for expansion: (i) gastric/gastroesophageal junction tumor, intolerant to/progressed after first-line treatment, and no prior anti-programmed cell death receptor-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy; (ii) bladder/urothelial tumor, intolerant to/progressed after first-line cisplatin-based chemotherapy, and no prior anti-PD-1/PD-L1 therapy; (iii) non-small-cell lung cancer (NSCLC) with previous anti-PD-1/PD-L1 therapy. Patients received eprenetapopt 4.5 g/day intravenously (IV) on days 1-4 with pembrolizumab 200 mg IV on day 3 in each 21-day cycle. Primary endpoints were dose-limiting toxicity (DLT), adverse events (AEs), and recommended phase II dose (RP2D) of eprenetapopt. RESULTS: Forty patients were enrolled (median age 66 years; range 27-85) and 37 received eprenetapopt plus pembrolizumab. No DLTs were reported and the RP2D for eprenetapopt in combination was 4.5 g/day IV on days 1-4. The most common eprenetapopt-related AEs were dizziness (35.1%), nausea (32.4%), and vomiting (29.7%). AEs leading to eprenetapopt discontinuation occurred in 2/37 patients (5.4%). In efficacy-assessable patients (n = 29), one achieved complete response (urothelial cancer), two achieved partial responses (NSCLC, urothelial cancer), and six patients had stable disease. CONCLUSIONS: The eprenetapopt plus pembrolizumab combination was well tolerated with an acceptable safety profile and showed clinical activity in patients with solid tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Persona de Mediana Edad , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Quinuclidinas/uso terapéuticoRESUMEN
Expression of the DNA repair and recombination protein human Rad51 (HsRad51) is increased in transformed cells and in cancer cell lines. In order to study the effects of acute HsRad51 ectopic overexpression on cell proliferation, cell cycle progression, and apoptosis, we generated clones of the human fibrosarcoma cell line HT1080 carrying a HsRad51 transgene under a repressible promoter. The HsRad51-overexpressing cells showed decreased plating efficiency and growth rate in a dose-dependent manner with regard to the degree of overexpression. An accumulation of HsRad51-overexpressing cells in G(2) was observed following release of cells after synchronization with double thymidine block. Moreover, the fraction of apoptotic cells measured by annexin V-FACS increased with the time of HsRad51 overexpression. In the light of these observations, sustained increased levels of HsRad51 may contribute to tumor progression by causing a selection for cells tolerant to the growth-suppressive and apoptosis-inducing effects of acute HsRad51 overexpression.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/biosíntesis , Expresión Génica , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Células Clonales/citología , Células Clonales/metabolismo , Ensayo Cometa , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Fibrosarcoma/metabolismo , Citometría de Flujo , Humanos , Regiones Promotoras Genéticas , Recombinasa Rad51 , Transgenes , Ensayo de Tumor de Célula MadreRESUMEN
OBJECTIVES: To analyze the early gene expression in macrophages accompanying the phenotypic changes into foam cells upon exposure to oxidized low-density lipoprotein. To identify candidate genes and markers for further studies into the pathogenesis of atherosclerosis. METHODS: Cells of the monocytic cell line THP-1 were activated by PMA and exposed to oxidized low-density lipoprotein. Gene expression profiles were investigated after 24 h, using a solid phase cDNA representational difference analysis (RDA) method and shotgun sequencing. Results were verified by microarray hybridization, and analyzed in the virtual chip display of a novel software tool for transcript profile exploration. RESULTS: By comparing transcript profiles of exposed/unexposed cells, 1,984 transcript sequences, representing a total of 921 genes with altered expression levels in response to oxidized low-density lipoprotein exposure, were identified. Genes that are central to cell cycle control and proliferation, inflammatory response, and of pathways not previously implicated in atherosclerosis were identified. The data obtained is also made available on-line at http:// biobase.biotech.kth.se/thp1a for further exploration. CONCLUSION: The identification of new candidate genes for atherosclerotic disease through RDA-based transcript profiling facilitates further functional genomic studies in coronary artery disease. Candidate genetic polymorphism markers of potential clinical relevance can be identified by filtering information in genome variation databases through the virtual chip analysis of the transcript profiles and subsequently tested in association studies.
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Arteriosclerosis/genética , Células Espumosas/metabolismo , Perfilación de la Expresión Génica , Lipoproteínas LDL/metabolismo , Línea Celular , ADN/análisis , Células Espumosas/patología , Regulación de la Expresión Génica , Humanos , Activación de Macrófagos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.
Asunto(s)
Caspasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Secuencia de Aminoácidos , Apoptosis/fisiología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Caspasa 3 , Inhibidores de Caspasas , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Células Jurkat/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/análisis , Recombinasa Rad51 , Proteínas Recombinantes de Fusión/metabolismo , Estaurosporina/farmacología , Especificidad por Sustrato , Células Tumorales Cultivadas/metabolismoRESUMEN
Constant denaturant capillary electrophoresis (CDCE), based on co-operative DNA melting equilibria, has the resolving power to separate single nucleotide mutants from wild type sequences. We used this technique to study mutations in a 70-bp isomelting domain of the human HPRT gene, which included the entire exon 5 and its flanking splice donor and acceptor sites. Pooled samples of 6-thioguanine selected T-cell clones from 51 healthy donors representing a total of approximately 1000 individual HPRT mutants were analysed. Slow moving peaks from the heteroduplex part of the CDCE electropherograph were collected and subjected to a second round of PCR and CDCE analysis, followed by DNA sequencing. Five independent mutations were detected. Four were splicing errors; one insertion of CC and two G-->A transitions in the splice donor site of intron 5, and one G-->C transversion in the splice acceptor site of intron 4. The fifth mutation was a missense transversion, T389>G. A reconstruction experiment, in which DNA with known mutation was mixed with wild type DNA, showed the sensitivity of mutation detection to be better than 1:100 under the conditions used in this study. These results demonstrate the high sensitivity of the CDCE-method for mutation screening.
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Exones/genética , Hipoxantina Fosforribosiltransferasa/genética , Linfocitos T/enzimología , Secuencia de Bases , ADN/química , ADN/genética , Análisis Mutacional de ADN , Electroforesis Capilar/métodos , Humanos , Masculino , Mutación , Desnaturalización de Ácido Nucleico , Mutación Puntual , Sensibilidad y Especificidad , Linfocitos T/citología , TemperaturaRESUMEN
A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a "virtual chip," in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.
Asunto(s)
Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Programas Informáticos , Transcripción Genética , Presentación de Datos , Genes , Humanos , Internet , Neoplasias/genéticaRESUMEN
Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G0-lymphocytes exposed to gamma-irradiation at both high dose rate (45 Gy h-1) and low dose rate (0.024 Gy h-1) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G0 after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.
Asunto(s)
Aberraciones Cromosómicas , Rayos gamma , Linfocitos T/efectos de la radiación , Expansión de Repetición de Trinucleótido/genética , Células Clonales , Relación Dosis-Respuesta en la Radiación , Variación Genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Cariotipificación , Tolerancia a RadiaciónRESUMEN
The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Linfocitos T/enzimología , Anticuerpos Monoclonales , Camptotecina/farmacología , Línea Celular Transformada , Sistema Libre de Células , Inhibidores de Cisteína Proteinasa/farmacología , ADN-Topoisomerasas de Tipo I/farmacología , Proteína Quinasa Activada por ADN , Humanos , Células Jurkat/enzimología , Proteínas Nucleares , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasa Rad51 , Inhibidores de Serina Proteinasa/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Receptor fas/fisiologíaRESUMEN
The use of algorithms for calculation and analysis of RNA secondary structures has largely been limited to mainframe computers, mainly due to the 16-bit memory restrictions imposed by MS-DOS. The program presented here, RNAdraw, moves calculations to the 32-bit Microsoft Windows environments with an intuitive user interface with extensive viewing, editing and printing possibilities. The algorithms for secondary structure/basepair probability matrix/heat curve calculation have been ported directly from a 32-bit Unix environment. RNAdraw also offers novel features such as the options to edit energy parameters, extract structures of different probability levels, create de novo secondary structures interactively, and combine viewing of structures and basepair probabilities.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Programas Informáticos , Algoritmos , Secuencia de Bases , Redes de Comunicación de Computadores , Gráficos por Computador , Simulación por Computador , Microcomputadores , Modelos Moleculares , Datos de Secuencia Molecular , ARN/genética , TermodinámicaRESUMEN
The effect of heat-shock on the expression of c-myc genes in different chromosomal contexts was investigated in a panel of human B-lymphoid cell lines. Burkitt's lymphoma cell lines with c-myc translocation breakpoints upstream of the first exon, within the exon itself, or in the first intron showed downregulation of c-myc levels as did a cell line without any translocation. The c-myc mRNA of cell lines with translocation breakpoints within the c-myc gene have previously been reported to have prolonged half-lives. After heat shock, the levels of these mRNA species were reduced with similar kinetics as the normal c-myc mRNA. An exception was an RNA species where the only c-myc sequences are derived from exon 1, showing that sequences from this part of the c-myc gene are not sufficient to mediate the rapid downregulation. Nuclear run-on analysis did not show reduced transcription of c-myc after heat shock and a comparison of cytoplasmic and total RNA did not indicate accumulation of longer, unspliced c-myc mRNA species. These observations suggest a posttranscriptional, cytoplasmic downregulation targeting exons 2 and/or 3. B-lymphoma lines transfected with a hsp70 promoter-linked c-myc gene were deficient in their ability to reinitiate proliferation after heat shock, providing a physiological rationale for the normal downregulation of c-myc after this type of physical stress.
Asunto(s)
Linfocitos B/metabolismo , Regulación hacia Abajo/fisiología , Genes myc/fisiología , Calor , ARN Mensajero/biosíntesis , Secuencia de Bases , Northern Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Humanos , Linfoma/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional/fisiología , Transfección , Células Tumorales CultivadasRESUMEN
We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.
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Endorribonucleasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Evolución Biológica , Línea Celular , Reacciones Cruzadas , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Escherichia coli/enzimología , Genes myc , Humanos , Datos de Secuencia Molecular , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Procesamiento Postranscripcional del ARN , Especificidad de la Especie , Especificidad por SustratoRESUMEN
We have demonstrated previously that overproduction of c-Myc, N-Myc and, to a lesser extent, L-Myc facilitates the replication of simian virus 40 (SV40)-based vectors in human lymphoid cells. Using a series of c-myc deletion mutants, we investigated which c-Myc regions are important in stimulating SV40 replication. The ability of c-Myc to promote SV40 replication was significantly reduced by deletions in the second exon domain, formerly shown to be crucial for c-Myc's transforming capacity. The c-myc mutants with a disrupted basic region (b) or leucine zipper (Zip) motif were also unable to stimulate SV40 replication. These regions are implicated in protein-DNA and protein-protein interactions, respectively, suggesting that the c-Myc protein might be associated with the DNA-protein replication complex. We present data obtained from gel mobility shift assays and from an immunocomplex-binding assay substantiating this hypothesis.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/fisiología , Virus 40 de los Simios/fisiología , Replicación Viral/fisiología , Secuencia de Bases , Northern Blotting , Western Blotting , ADN Viral , Técnica del Anticuerpo Fluorescente , Genes myc , Humanos , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-myc/genética , Mapeo Restrictivo , Eliminación de Secuencia , Virus 40 de los Simios/genética , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Chromosome rearrangements involving band 19p13 have been described in about half of all ovarian carcinomas. Four ovarian carcinomas with translocations of 19p13 were investigated with 11 DNA probes for markers localized to this band. All markers exhibited normal restriction patterns, suggesting that they were not rearranged. The 19p13 breakpoint of one tumor was mapped to a location between the INSR and the TCF3 loci.
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Carcinoma/genética , Mapeo Cromosómico , Cromosomas Humanos Par 19 , Neoplasias Ováricas/genética , Translocación Genética , Sondas de ADN , ADN de Neoplasias/análisis , Femenino , Marcadores Genéticos , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Receptor de Insulina/genética , Factores de Transcripción/genéticaRESUMEN
The levels of c-myc protein expression in three types of Epstein-Barr virus (EBV) transformed human B-cell derived lines were examined with an ELISA assay. Six independently maintained sublines of the same EBV-transformed pro-B-cell line (FLEB-14), six B-cell lines (LCL) and six Burkitt's lymphoma lines (BL) were compared. The average amount of c-myc protein, calculated from at least three independent tests on each line, differed between the three groups. Expressed in relative units, the ratio of the means was 1:2:5 for the LCL:FLEB:BL lines. The differences were statistically significant at P less than 0.01.
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Linfoma de Burkitt/metabolismo , Proteínas Proto-Oncogénicas c-myc/análisis , Linfocitos B/metabolismo , Linfoma de Burkitt/genética , Línea Celular , Transformación Celular Viral/fisiología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Regulación Viral de la Expresión Génica , HumanosRESUMEN
We have determined the phenotype of EBV-carrying cells in human blood by establishing spontaneous LCLs from populations selected according to three B cell markers, B1, B2, and 35.1C5. LCLs grew from the B1-positive, B2-positive, B2-negative and 35.1C5-positive population. Thus, EBV-carrying cells in blood of seropositive individuals are restricted to the B cell lineage. These B cells may or may not express the B2 epitope, and they may be derived from the lymph node mantle zone or the splenic marginal zone. These in vivo EBV-carrying cells could enter the viral productive cycle and transform coresident B cells during the initial 3 days in vitro. The characteristic of the LCLs with regard to these 3 B cell markers did not correspond to the original B cell population from which they were derived.
Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Antígenos de Superficie , Linfocitos B/microbiología , Herpesvirus Humano 4/inmunología , Adulto , Linfocitos B/inmunología , Biomarcadores , Línea Celular , Herpesvirus Humano 4/aislamiento & purificación , HumanosRESUMEN
The steady-state level of c-myc proto-oncogene mRNA was investigated in the EBV-negative human B-lymphoma line BJAB and 2 sublines that have been converted by EBV into stable EBV-genome-carrying and EBNA-positive status. The EBV-converted sublines expressed c-myc at a 2- to 6-fold higher level than the original BJAB during exponential growth. The EBV-positive BJAB lines are known to differ from the parent line in several phenotypic characteristics, including increased agarose clonability, lower serum requirement and, in one case, increased tumorigenicity in nude mice. The pattern of increased c-myc expression accompanying EBV conversion was not observed in the myc/Ig translocation-carrying Burkitt lymphoma line RAMOS or in 2 of its EBV-converted sublines.