RESUMEN
The functions of androgen receptor (AR) in stromal cells are still debated in spite of the demonstrated importance of these cells in organ development and diseases. Here, we show that physiological androgen concentration (10 nM R1881 or DHT) fails to induce DNA synthesis, while it consistently stimulates cell migration in mesenchymal and transformed mesenchymal cells. Ten nanomolar R1881 triggers p27 Ser10 phosphorylation and its stabilization in NIH3T3 fibroblasts. Activation of Rac and its downstream effector DYRK 1B is responsible for p27 Ser10 phosphorylation and cell quiescence. Ten nanomolar androgen also inhibits transformation induced by oncogenic Ras in NIH3T3 fibroblasts. Overexpression of an AR mutant unable to interact with filamin A, use of a small peptide displacing AR/filamin A interaction, and filamin A knockdown indicate that the androgen-triggered AR/filamin A complex regulates the pathway leading to p27 Ser10 phosphorylation and cell cycle arrest. As the AR/filamin A complex is also responsible for migration stimulated by 10 nM androgen, our report shows that the androgen-triggered AR/filamin A complex controls, through Rac 1, the decision of cells to halt cell cycle and migration. This study reveals a new and unexpected role of androgen/AR signalling in coordinating stromal cell functions.
Asunto(s)
Dihidrotestosterona/farmacología , Filaminas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Filaminas/genética , Regulación de la Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Metribolona/farmacología , Ratones , Células 3T3 NIH , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores Androgénicos/genética , Serina/metabolismo , Congéneres de la Testosterona/farmacología , Células Tumorales Cultivadas , Proteína de Unión al GTP rac1/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Quinasas DyrKRESUMEN
Nanog is a transcription factor required for maintaining the pluripotency of embryonic stem cells, and is not expressed in most normal adult tissues. However, recent studies have indicated that Nanog is overexpressed in many types of human cancers, including breast cancer. To elucidate the physiological roles of Nanog in tumorigenesis, we developed an inducible Nanog transgenic mouse model, in which the expression of Nanog in adult tissues can be induced via LoxP/Cre-mediated deletion. Our findings indicate that overexpression of Nanog in the mammary gland is not sufficient to induce mammary tumor. However, when coexpressed with Wnt-1 in the mouse mammary gland, it promotes mammary tumorigenesis and metastasis. In this context, Nanog promotes the migration and invasion of breast cancer cells. Microarray analysis has shown that the ectopic expression of Nanog deregulates the expression of numerous genes associated with tumorigenesis and metastasis, such as the PDGFRα gene. Our findings demonstrate the involvement of Nanog in breast cancer metastasis, and provide the basis for the reported correlation between Nanog expression and poor prognosis of human breast cancer patients. As Nanog is not expressed in most adult tissues, these findings identify Nanog as a potential therapeutic target in the treatment of Nanog-expressing metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Proteínas de Homeodominio/fisiología , Neoplasias Mamarias Animales/fisiopatología , Metástasis de la Neoplasia , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cartilla de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Proteína Homeótica Nanog , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In response to stress, p53 is accumulated and activated to induce appropriate growth inhibitory responses. This requires the release of p53 from the constraints of its negative regulators Mdm2 and Mdm4. A key event in this dissociation is the phosphorylation of p53 at threonine residue (Thr18) within the Mdm2/4-binding domain. Casein kinase 1 (CK1) plays a major role in this phosphorylation. The promyelocytic leukemia protein (PML) regulates certain modifications of p53 in response to DNA damage. Here, we investigated the role of PML in the regulation of Thr18 phosphorylation. We found that PML enhances Thr18 phosphorylation of endogenous p53 in response to stress. On DNA damage, CK1 accumulates in the cell, with a proportion concentrated in the nucleus together with p53 and PML. Furthermore, CK1 interacts with endogenous p53 and PML, and this interaction is enhanced by genotoxic stress. Inhibition of CK1 impairs the protection of p53 by PML from Mdm2-mediated degradation. Our findings support a role for PML in the regulation of p53 by CK1. We propose that following DNA damage, PML facilitates Thr18 phosphorylation by recruiting p53 and CK1 into PML nuclear bodies, thereby protecting p53 from inhibition by Mdm2, leading to p53 activation.
Asunto(s)
Quinasa de la Caseína I/fisiología , Daño del ADN , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Transporte Activo de Núcleo Celular , Trióxido de Arsénico , Arsenicales/farmacología , Línea Celular Tumoral , Humanos , Óxidos/farmacología , Fosforilación , Proteína de la Leucemia Promielocítica , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Treonina/metabolismoRESUMEN
In human mammary and prostate cancer cells, steroid hormones or epidermal growth factor (EGF) trigger association of the androgen receptor (AR)-estradiol receptor (ER) (alpha or beta) complex with Src. This interaction activates Src and affects the G1 to S cell cycle progression. In this report, we identify the sequence responsible for the AR/Src interaction and describe a 10 amino-acid peptide that inhibits this interaction. Treatment of the human prostate or mammary cancer cells (LNCaP or MCF-7, respectively) with nanomolar concentrations of this peptide inhibits the androgen- or estradiol-induced association between the AR or the ER and Src the Src/Erk pathway activation, cyclin D1 expression and DNA synthesis, without interfering in the receptor-dependent transcriptional activity. Similarly, the peptide prevents the S phase entry of LNCaP and MCF-7 cells treated with EGF as well as mouse embryo fibroblasts stimulated with androgen or EGF. Interestingly, the peptide does not inhibit the S phase entry and cytoskeletal changes induced by EGF or serum treatment of AR-negative prostate cancer cell lines. The peptide is the first example of a specific inhibitor of steroid receptor-dependent signal transducing activity. The importance of these results is highlighted by the finding that the peptide strongly inhibits the growth of LNCaP xenografts established in nude mice.
Asunto(s)
Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores Androgénicos/metabolismo , Dominios Homologos src/fisiología , Secuencia de Aminoácidos , Antagonistas de Receptores Androgénicos , Animales , Neoplasias de la Mama/metabolismo , Humanos , Masculino , Ratones , Péptidos , Neoplasias de la Próstata/metabolismo , Unión Proteica , Receptores de Estradiol/antagonistas & inhibidores , Receptores de Estradiol/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
There is increasing evidence for the role of wild-type p53 induced phosphatase 1 (Wip1) phosphatase in the regulation of tumorigenesis. To evaluate Wip1 as a breast cancer oncogene, we generated a mouse strain with targeted expression of Wip1 to the breast epithelium. We found that these mice are prone to cancer when intercrossed with transgenics expressing the ErbB2 oncogene but not conditional knockouts for Brca2. This tumor-prone phenotype of Wip1 is fully eliminated through attenuation of proliferation by activating the MKK6/p38 mitogen-activated protein kinases (MAPK) cascade in mice bearing a constitutively active form of MKK6. We propose that Wip1 phosphatase operates within the MKK6/p38 MAPK signaling pathway to promote ErbB2-driven mammary gland tumorigenesis.
Asunto(s)
MAP Quinasa Quinasa 6/fisiología , Neoplasias Mamarias Experimentales/enzimología , Proteínas de Neoplasias/fisiología , Fosfoproteínas Fosfatasas/fisiología , Receptor ErbB-2/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Fosfoproteínas Fosfatasas/deficiencia , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1 , Proteína Fosfatasa 2CRESUMEN
Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.
Asunto(s)
Membrana Celular/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Biotinilación , Cristalografía por Rayos X , Dimerización , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fenotipo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Linfocitos T Citotóxicos/metabolismo , Factores de TiempoRESUMEN
Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.
Asunto(s)
Melanosomas/química , Proteínas de Neoplasias/análisis , Oxidorreductasas , Electroforesis/métodos , Humanos , Oxidorreductasas Intramoleculares/análisis , Melanosomas/ultraestructura , Glicoproteínas de Membrana/análisis , Microscopía Confocal/métodos , Microscopía Inmunoelectrónica/métodos , Monofenol Monooxigenasa/análisis , Proteínas/análisis , Células Tumorales Cultivadas , Antígeno gp100 del MelanomaRESUMEN
The development of effective T cell-based immunotherapy for cancer requires the identification of antigens capable of inducing both CTL and T helper immune responses. Although CTLs will participate in the antitumor response mainly by exerting their lytic activity on the tumor cells, helper T lymphocytes will be critical for the induction and maintenance of the CTLs. Thus, effective subunit therapeutic vaccines should include both CTL and T helper epitopes from antigens expressed on the tumor cells. The product of the MAGE-A3 gene is an attractive candidate for tumor immunotherapy because it is expressed in the majority of melanomas and in a great proportion of other solid tumors. Although numerous CTL epitopes for the MAGE-A3 antigen have been reported, only a few have been described for helper T cells. Here we show that a synthetic peptide derived from the MAGE-A3 sequence (MAGE-A3(146-160)) was effective in inducing in vitro T helper responses in the context of HLA-DR4 and HLA-DR7 alleles. Most significantly, the peptide-reactive helper T lymphocytes were capable of recognizing various forms of MAGE-A3 antigen (tumor cell lysates, dead/apoptotic tumor cells, or recombinant MAGE-A3 protein), indicating that the T-cell epitope represented by peptide MAGE-A3(146-160) is naturally processed by antigen-presenting cells. These studies are relevant for the design of multi-epitope vaccines for treating MAGE-A3-expressing tumors through the simultaneous stimulation of CTL and T helper lymphocytes.
Asunto(s)
Antígenos de Neoplasias/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-DR4/inmunología , Antígeno HLA-DR7/inmunología , Proteínas de Neoplasias , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Humanos , Células L , Activación de Linfocitos/inmunología , Melanoma/inmunología , Ratones , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8-10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these "Trojan" Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Presentación de Antígeno , Epítopos de Linfocito T/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Antígeno Carcinoembrionario/inmunología , Antígeno Carcinoembrionario/metabolismo , Línea Celular , Proteínas del Huevo/genética , Proteínas del Huevo/inmunología , Proteínas del Huevo/metabolismo , Epítopos de Linfocito T/inmunología , Productos del Gen tat/síntesis química , Productos del Gen tat/genética , Productos del Gen tat/inmunología , Productos del Gen tat/metabolismo , Vectores Genéticos/inmunología , Vectores Genéticos/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fragmentos de Péptidos/síntesis química , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/inmunología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Citotóxicos/enzimología , Células Tumorales CultivadasRESUMEN
In unstressed cells, the tumor suppressor protein p53 is present in a latent state and is maintained at low levels through targeted degradation. A variety of genotoxic stresses initiate signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. Recent studies point to roles for multiple post-translational modifications in mediating these events in response to genotoxic stresses through several potentially interacting but distinct pathways. The approximately 100 amino-acid N-terminal and approximately 90 amino-acid C-terminal domains are highly modified by post-translational modifications. The N-terminus is heavily phosphorylated while the C-terminus contains phosphorylated, acetylated and sumoylated residues. Antibodies that recognize p53 only when it has been modified at specific sites have been developed, and studies with these reagents show that most known post-translational modifications are induced when cells are exposed to genotoxic stresses. These recent results, coupled with biochemical and genetic studies, suggest that N-terminal phosphorylations are important for stabilizing p53 and are crucial for acetylation of C-terminal sites, which in combination lead to the full p53-mediated response to genotoxic stresses. Modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding; additionally, they modulate the stability, the oligomerization state, the nuclear import/export process and the degree of ubiquitination of p53.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Animales , Sitios de Unión , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/químicaRESUMEN
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3 , Animales , Fase G2/genética , Células HeLa , Humanos , Ratones , Mitosis/genética , Índice Mitótico , Fosforilación , Unión Proteica , Serina/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.
Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Antígenos HLA-DR/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular , Reacciones Cruzadas , Mapeo Epitopo , Infecciones por VIH/inmunología , Humanos , Datos de Secuencia Molecular , Péptidos/inmunologíaRESUMEN
The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Genes Supresores de Tumor , Humanos , Proteínas Proto-Oncogénicas c-mdm2 , Supresión Genética , Proteína p14ARF Supresora de TumorRESUMEN
We have developed efficient methods for epitope identification and vaccine design. Our process for epitope selection based on the combined use of motif analyses, binding assays and immunogenicity evaluations is described. We also describe how the projected population coverage and vaccine design can be optimized. Finally, it is discussed how vaccine potency is evaluated by immunogenicity and antigenicity assays.
Asunto(s)
Epítopos/inmunología , Vacunas/inmunología , Vacunas/química , Vacunas/uso terapéuticoRESUMEN
Single amino acid substitution analogs of the known Mamu A*01 binding peptide gag 181-190 and libraries of naturally occurring sequences of viral or bacterial origin were used to rigorously define the peptide binding motif associated with Mamu A*01 molecules. The presence of S or T in position 2, P in position 3, and hydrophobic or aromatic residues at the C terminus is associated with optimal binding capacity. At each of these positions, additional residues are also tolerated but associated with significant decreases in binding capacity. The presence of at least two preferred and one tolerated residues at the three anchor positions is necessary for good Mamu A*01 binding; optimal ligand size is 8-9 residues. This detailed motif has been used to map potential epitopes from SIVmac239 regulatory proteins and to engineer peptides with increased binding capacity. A total of 13 wild type and 17 analog candidate epitopes were identified. Furthermore, our analysis reveals a significantly lower than expected frequency of epitopes in early regulatory proteins, suggesting a possible evolutionary- and/or immunoselection directed against variants of viral products that contain CTL epitopes.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Fragmentos de Péptidos/metabolismo , Virus de la Inmunodeficiencia de los Simios/inmunología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Algoritmos , Sustitución de Aminoácidos , Aminoácidos/metabolismo , Animales , Sitios de Unión/inmunología , Epítopos de Linfocito T/metabolismo , Proteínas Inmediatas-Precoces/síntesis química , Proteínas Inmediatas-Precoces/metabolismo , Ligandos , Macaca mulatta , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/síntesis química , Mapeo Peptídico , Unión Proteica/inmunología , Ingeniería de Proteínas , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/síntesis químicaRESUMEN
Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53(264-272) epitope to generate CTL from circulating precursor T cells of HLA-A2.1(+) healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53(264-272) peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53(264-272) peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53(264-272) epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53(264-272) epitope. These findings suggest that in vivo, CTL specific for the wt p53(264-272) peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Epítopos de Linfocito T/metabolismo , Neoplasias de Cabeza y Cuello/inmunología , Epítopos Inmunodominantes/metabolismo , Activación de Linfocitos/genética , Fragmentos de Péptidos/metabolismo , Subgrupos de Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/metabolismo , Autoanticuerpos/sangre , Carcinoma de Células Escamosas/genética , Citotoxicidad Inmunológica/genética , Análisis Mutacional de ADN , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Citometría de Flujo , Variación Genética/inmunología , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Neoplasias de Cabeza y Cuello/genética , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Coloración y Etiquetado , Subgrupos de Linfocitos T/química , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/química , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
It is accepted that both helper and CTLs play a critical role in immune antitumor responses. Thus, the design of effective immune-based therapies for cancer relies in the identification of relevant tumor-associated antigens (TAAs) capable of eliciting strong helper and cytotoxic T-cell responses against tumor cells. The product of the HER2/neu oncogene is considered as a prototype TAA, because it is found overexpressed in a large variety of malignancies, whereas normal cells only produce low levels of this product. Several cytotoxic T-cell epitopes for HER2/neu have been identified that enable the design of peptide-based therapeutic vaccines for tumors expressing this TAA. Nevertheless, it is expected that inclusion of peptide epitopes capable of eliciting HER2/neu-specific T helper responses into these vaccines may enhance their effectiveness in the clinic. We describe here a strategy to identify helper T-cell epitopes for HER2/neu that focuses on peptides predicted to bind to numerous histocompatibility alleles (promiscuous epitopes), which would encourage their use in therapeutic vaccines for the general cancer patient population. Following this approach, we successfully identified several peptides that elicited T helper (CD4+) proliferative responses to peptides derived from HER2/neu. Most of the T-cell responses appeared to reflect a low affinity for antigen, which could be the result of immune tolerance because HER2/neu is expressed in low levels in normal cells and possibly including lymphocytes and monocytes. Interestingly, one of these peptides, HER2(883), was recognized by T cells in the context of either HLA-DR1, HLA-DR4, HLA-DR52, and HLA-DR53, indicating a high degree of histocompatibility promiscuity. Furthermore, T cells that reacted with peptide HER2(883) could also recognize antigen-presenting cells that process HER2/neu recombinant protein. These results may be relevant for the design of more effective therapeutic vaccines for tumors expressing the HER2/neu oncogene product.
Asunto(s)
Antígenos de Neoplasias/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/inmunología , Receptor ErbB-2/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/sangre , Linfocitos T CD4-Positivos/inmunología , Línea Celular Transformada , Mapeo Epitopo , Antígenos HLA-DR/genética , Humanos , Células L , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/sangre , TransfecciónRESUMEN
Ser-15 of human p53 (corresponding to Ser-18 of mouse p53) is phosphorylated by ataxia-telangiectasia mutated (ATM) family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser-18 on biological responses to DNA damage, we introduced a missense mutation (Ser-18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser-18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser-18 was not required for ATM-dependent cellular resistance after exposure to IR. Additionally, efficient acetylation of the C terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser-18.