RESUMO
Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Hemofilia A/genética , Hemofilia A/terapia , Adulto , Reações Cruzadas/imunologia , Dependovirus/imunologia , Fator VIII/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Humoral , Masculino , Transgenes , Resultado do TratamentoRESUMO
CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proteína-1 Reguladora de Fusão/antagonistas & inibidores , Aminoácidos/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Transporte Biológico , Caspases/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: We investigated Epstein-Barr virus (EBV) antibody kinetics in university freshmen who developed laboratory-documented primary EBV infection during prospective studies and correlated these kinetics with disease severity. METHODS: EBV-naïve participants had blood collected periodically and sera tested for EBV-specific antibodies with line blot and enzyme immunoassays. The line blot assay contained EBNA-1, p18, p23, BZLF-1, p138, and p54 antigens; the enzyme immunoassay contained viral capsid antigen and EBNA-1. Severity of illness (SOI) was graded 0 (asymptomatic) to 6 (bedridden). Participants with maximum SOI scores 0-2 were compared with those whose maximum SOI scores were 3-6. Time to first antibody response was analyzed using the semi-parametric COX model. RESULTS: A total of 201 sera from 38 college students collected before, during, and after primary EBV infection were tested. Earlier antibody responses correlated with milder symptoms. This was most pronounced for late-developing antibodies. The median time to development of p18 IgG was significantly earlier among low-SOI participants (64 days) than high-SOI patients (119 days; P = 0.0003).). Participants with mild disease developed EBNA-1 antibodies sooner than participants with more severe disease (125 days versus >270 days; P = 0.017). Participants with mild disease also showed more rapid loss of antibodies against IgG EA p138 and p54 ≥12 weeks post-infection (P = 0.012 and P = 0.026, respectively). CONCLUSIONS: These data suggest that rapid antibody responses to EBV correlate with reduced severity of primary EBV infection.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Anticorpos Antivirais , Formação de Anticorpos , Antígenos Virais , Infecções por Vírus Epstein-Barr/diagnóstico , Humanos , Imunoglobulina G , Estudos ProspectivosRESUMO
No licensed vaccine is available for prevention of EBV-associated diseases, and robust, high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed an improved EBV-GFP based neutralization assay for such a vaccine's pre-clinical and clinical validation to measure EBV specific neutralizing antibodies in human donors. The supplementation of guinea pig complement of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay allowed the detection of complement-dependent neutralizing antibodies using a panel of heat-inactivated healthy human sera. Anti-gp350 antibody titers, which were evaluated using a previously optimized anti-gp350 IgG ELISA assay, were moderately correlated to the FFA-based neutralization titers. Overall, this improved high-throughput neutralization assay is capable of characterizing the serologic neutralizing antibody response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.
RESUMO
Aberrant patterns of pre-mRNA processing are typical of human malignancies, yet the mechanisms responsible for these changes remain undefined. We have recently shown overexpression of a core splice regulatory protein, serine-arginine protein kinase 1 (SRPK1), in dysplastic and neoplastic pancreatic ductular cells. In the present study, we have established that SRPK1 levels are similarly up-regulated in breast and colonic tumors where its expression increases coordinately with tumor grade. Targeting SRPK1 for inhibition using small interfering RNA in breast and colonic tumor cell lines in vitro resulted in both increased apoptotic potential and enhanced cell killing after treatment with gemcitabine and cisplatin. Recent reports have described multifaceted interactions between the mitogen-activated protein kinase (MAPK) and AKT signaling networks and the splice regulatory machinery. Consequently, we have shown that targeted inhibition of SRPK1 in tumor cells results in reduced phosphorylation of MAPK3, MAPK1, and AKT. Alterations in the splice pattern and resulting expression of MAPK kinase are implicated in mediating the antitumoral effects resulting from SRPK1 down-regulation. The up-regulation of SRPK1 in multiple cancers and its ability to regulate multiple relevant signaling pathways provide support for developing agents to inhibit this kinase for possible broad application to treat epithelial cancers.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Processamento Alternativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Células CACO-2 , Carcinoma/enzimologia , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Células HT29 , Humanos , MAP Quinase Quinase 2/metabolismo , Neoplasias Pancreáticas/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transduction where antibodies are not detected. To better define the relationship between various forms of pre-existing AAV immunity and gene transfer, we studied valoctocogene roxaparvovec (BMN 270) in cynomolgus monkeys with varying pre-dose levels of neutralizing anti-AAV antibodies and non-antibody transduction inhibitors. BMN 270 is an AAV5-based vector for treating hemophilia A that encodes human B domain-deleted factor VIII (FVIII-SQ). After infusion of BMN 270 (6.0 × 1013 vg/kg) into animals with pre-existing anti-AAV5 antibodies, there was a mean decrease in maximal FVIII-SQ plasma concentration (Cmax) and AUC of 74.8% and 66.9%, respectively, compared with non-immune control animals, and vector genomes in the liver were reduced. In contrast, animals with only non-antibody transduction inhibitors showed FVIII-SQ plasma concentrations and liver vector copies comparable with those of controls. These results demonstrate that animals without AAV5 antibodies are likely responders to AAV5 gene therapy, regardless of other inhibiting plasma factors. The biological threshold for tolerable AAV5 antibody levels varied between individual animals and should be evaluated further in clinical studies.
RESUMO
Aberrant patterns of pre-mRNA splicing have been established for many human malignancies, yet the mechanisms responsible for these tumor-specific changes remain undefined and represent a promising area for therapeutic intervention. Using immunohistochemistry, we have localized the expression of a central splicing regulator, serine-arginine protein kinase 1 (SRPK1), to the ductular epithelial cells within human pancreas and have further shown its increased expression in tumors of the pancreas, breast, and colon. Small interfering RNA-mediated down-regulation of SRPK1 in pancreatic tumor cell lines resulted in a dose-dependent decrease in proliferative capacity and increase in apoptotic potential. Coordinately, the disruption of SRPK1 expression resulted in enhanced sensitivity of tumor cells to killing by gemcitabine and/or cisplatin. A dose-dependent reduction in the phosphorylation status of specific SR proteins was detected following the down-regulation of SRPK1 and is likely responsible for the observed alterations in expression of proteins associated with apoptosis and multidrug resistance. These data support SRPK1 as a new, potential target for the treatment of pancreatic ductular cancer that at present remains largely unresponsive to conventional therapies. Furthermore, these results support the development of innovative therapies that target not only specific splice variants arising during tumorigenesis but also the splice regulatory machinery that itself may be abnormal in malignant cells.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Carcinoma Ductal Pancreático/enzimologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Regulação para Baixo , Humanos , Neoplasias Pancreáticas/enzimologia , Fosforilação , Proteínas Serina-Treonina Quinases/biossíntese , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transfecção , GencitabinaRESUMO
Prospective studies of antibodies to multiple Epstein-Barr virus (EBV) proteins and EBV neutralizing antibodies in the same individuals before, during, and after primary EBV infection have not been reported. We studied antibody responses to EBV in college students who acquired primary EBV infection during prospective surveillance and correlated the kinetics of antibody response with the severity of disease. Neutralizing antibodies and enzyme-linked immunosorbent assay (ELISA) antibodies to gp350, the major target of neutralizing antibody, reached peak levels at medians of 179 and 333 days after the onset of symptoms of infectious mononucleosis, respectively. No clear correlation was found between the severity of the symptoms of infectious mononucleosis and the peak levels of antibody to individual viral proteins or to neutralizing antibody. In summary, we found that titers of neutralizing antibody and antibodies to multiple EBV proteins increase over many months after primary infection with EBV.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Herpesvirus Humano 4/imunologia , Mononucleose Infecciosa/imunologia , Imunidade Adaptativa , Ensaio de Imunoadsorção Enzimática , Humanos , Mononucleose Infecciosa/patologia , Testes de Neutralização , Estudos Prospectivos , Índice de Gravidade de Doença , Estudantes , Fatores de TempoRESUMO
Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Mapeamento de Epitopos , Herpesvirus Humano 4/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linfócitos B/imunologia , Células CHO , Cricetulus , Escherichia coli , Glicosilação , Herpesvirus Humano 4/genética , Ligação Proteica , Conformação Proteica , Coelhos , Receptores de Complemento 3d/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Soft tissue sarcoma (STS) is a heterogenous tumor arising from the embryonic mesoderm represented by approximately 50 histological subtypes. Effective therapeutic intervention is lacking for recurrent, late stage and metastatic disease. CD39, a cell-surface ectonucleotidase, has previously been shown to be upregulated in hematological malignancies and various epithelial tumors, but not in STS. Here, we show by mass spectrometry and immunohistochemistry that CD39 is highly expressed in primary patient sarcoma samples. Moreover, CD39 nucleotidase activity is enhanced in fibrosarcoma compared with normal control cells. We demonstrate that an inhibitory monoclonal anti-CD39 antibody, abrogates CD39 enzymatic activity significantly and prolongs survival in a lethal metastatic patient-derived sarcoma model. Taken together, the data suggest CD39 is a novel therapeutic target for the treatment of STS.
RESUMO
Previous studies have suggested that differences in the ability of normal and malignant cells to process certain alternatively spliced pre-mRNA transcripts can be exploited as a potentially powerful means of targeting the expression of therapeutic genes to tumor cells in vivo and in vitro. Specifically, it was shown that efficient processing of minigene constructs containing the alternatively spliced CD44 exons v9 and v10 only occurs in tumor cells that express CD44 isoforms that incorporate these exons (e.g. CD44R1). In the present study, efforts were made to define the molecular mechanisms that underlie the apparent specificity of this process. RT-PCR analysis and DNA sequencing were used to characterize the various splicing events that occur between CD44 exons v8, v9 and v10 following transfection of minigene constructs containing these various exons into CD44R1-positive (PC3) and CD44R1-negative (T24) cell lines. The results obtained confirm that although the v8-v9 intron is efficiently removed in both CD44R1-positive and CD44R1-negative cells, the corresponding v9-v10 intron is accurately spliced and the exons appropriately joined only in lines that express v10-containing CD44 isoforms (e.g. PC3). In CD44R1-negative cell lines (e.g. T24) alternative 5' and 3' splice sites located within the v9-v10 intron are preferentially used, resulting in various portions of the intron being retained within the final processed mRNA product. It is proposed that identification of these functionally important intronic sequence elements will facilitate the development of second generation "splice activated gene expression" vectors that may prove useful in various cancer gene therapy applications.
Assuntos
Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Receptores de Hialuronatos/genética , Neoplasias/terapia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Vetores Genéticos/genética , Receptores de Hialuronatos/metabolismo , Íntrons/genética , Dados de Sequência Molecular , Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transfecção , Células Tumorais CultivadasRESUMO
In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid-based "splice-activated gene expression" (pSAGE) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a "leaderless" human liver/bone/kidney alkaline phosphatase (ALP) cDNA. Because both the v8-v9 and v9-v10 introns contain multiple in-frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre-mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H-positive (T24) and CD44R1-positive (PC3) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP/INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP-mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8-v9 intron is spliced in both CD44H- and CD44R1-positive cells, the v9-v10 intron is efficiently and accurately removed only in CD44R1-positive cells. Furthermore, only CD44R1-positive cells were sensitized to etoposide phosphate when transfected with the v9-v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.
Assuntos
Processamento Alternativo , Carcinoma de Células de Transição/terapia , Regulação Neoplásica da Expressão Gênica/genética , Terapia Genética , Receptores de Hialuronatos/genética , Neoplasias da Próstata/terapia , Neoplasias da Bexiga Urinária/terapia , Fosfatase Alcalina/metabolismo , Antineoplásicos/uso terapêutico , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA/química , Éxons , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/metabolismo , Íntrons , Masculino , Plasmídeos , Reação em Cadeia da Polimerase , Neoplasias da Próstata/genética , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
PURPOSE: Prostate cancer is detected with increasing frequency but has a highly variable natural history and prognosis and active surveillance of men with low-risk prostate cancer would benefit greatly from minimally invasive methods to identify progression. We describe here two novel in vivo metrics of cell proliferation in men with prostate neoplasia. EXPERIMENTAL DESIGN: Three groups of men drank heavy water, a nonradioactive, stable isotopic tracer for 14 to 28 days: (i) healthy men, (ii) men scheduled for transrectal core needle biopsy, and (iii) men scheduled for radical prostatectomy. Prostate epithelial cells (PEC) were isolated from ejaculated seminal fluid in all subjects. Histologically graded lesions were microdissected from tissue slides obtained from subjects undergoing surgery and proliferation rates were measured from isolated cells via mass spectrometry. RESULTS: Proliferation rates of seminal PEC in healthy men (0.10%-0.27%/d) were stable on repeat sampling. Rates above 0.34%/d were seen only in patients with cancer where rates increased progressively from normal tissue through benign prostate hyperplasia, prostate intraepithelial neoplasia, and tumor grades III and IV in all subjects. Seminal PEC kinetics correlated highly with the most proliferative microdissected region in each subject (r(2) = 0.94). CONCLUSIONS: Prostate cell proliferation can be measured in vivo from microdissected histopathology sections or noninvasively from seminal fluid where the latter reflects the most proliferative region of the gland. This approach may allow monitoring of progression in men with low-risk prostate cancer.
Assuntos
Proliferação de Células , Próstata/citologia , Próstata/patologia , Neoplasias da Próstata/patologia , Sêmen/citologia , Adulto , Idoso , Separação Celular , Óxido de Deutério , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Antígeno Ki-67/análise , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: Heavy water ((2)H(2)O) labelling of DNA enables the measurement of low-level cell proliferation in vivo, using gas chromatography/pyrolysis isotope ratio mass spectrometry (GC/P/IRMS), but the methodology has been too complex for widespread use. Here, we report a simplified method for measuring proliferation of malignant B cells in patients with chronic lymphocytic leukemia (CLL). DESIGN AND METHODS: Patients were labelled with (2)H(2)O for 6 weeks; blood samples were obtained at 0, 3, and 6 weeks during (2)H(2)O labelling and 9, 12, and 16 weeks thereafter. Bone marrow was sampled at week 6. Phlebotomy was performed at multiple, non-research clinical sites. CLL cells were isolated in a central laboratory, using a novel RosetteSep-based method; DNA labelling was analyzed by GC/P/IRMS. RESULTS: In 26 of 29 patients, CLL cell isolation resulted in > or =95% purity for malignant CD5+ B cells; in one patient, malignant cells expressed marginal levels of CD5, and in two others, further sorting of CD5hi malignant cells was required. Cell yields correlated with white blood cell counts and exceeded GC/P/IRMS requirements ( approximately 10(7) cells) >98% of the time; high-quality DNA labelling data were obtained. RosetteSep isolation achieved adequate CLL cell purity from bone marrow in only 64% of samples, but greatly reduced subsequent sort time for impure samples. CONCLUSION: This method enables clinical studies of CLL cell proliferation outside of research settings, using a shorter (2)H(2)O intake protocol, a minimal sampling protocol, and centralised sample processing. The CLL cell isolation protocol may also prove useful in other applications. (clinicaltrials.gov identifier: NCT00481858).
Assuntos
Linfócitos B/patologia , Proliferação de Células , Leucemia Linfocítica Crônica de Células B/patologia , Adulto , Idoso , Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Células da Medula Óssea/patologia , Separação Celular/métodos , Ensaios Clínicos como Assunto/métodos , Citodiagnóstico/métodos , DNA/análise , Óxido de Deutério/química , Eficiência , Feminino , Citometria de Fluxo/métodos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto/métodosRESUMO
Gastrointestinal peptide hormone receptors, like somatostatin receptors, are often overexpressed in human cancer, allowing receptor-targeted tumor imaging and therapy. A novel candidate for these applications is the secretin receptor recently identified in pancreatic and cholangiocellular carcinomas. In the present study, secretin receptors were assessed in a non-gastrointestinal tissue, the human lung. Non-small-cell lung cancers (n=26), small-cell lung cancers (n=10), bronchopulmonary carcinoid tumors (n=29), and non-neoplastic lung (n=46) were investigated for secretin receptor protein expression with in vitro receptor autoradiography, using (125)I-[Tyr(10)] rat secretin and for secretin receptor transcripts with RT-PCR. Secretin receptor protein expression was found in 62% of bronchopulmonary carcinoids in moderate to high density, in 12% of non-small cell lung cancers in low density, but not in small cell lung cancers. In tumors found to be secretin receptor positive by autoradiography, RT-PCR revealed transcripts for the wild-type secretin receptor and for novel secretin receptor splice variants. In the non-neoplastic lung, secretin receptor protein expression was observed in low density along the alveolar septa in direct tumor vicinity in cases of acute inflammation, but not in histologically normal lung. In the autoradiographically positive peritumoral lung, RT-PCR showed transcripts for the wild-type secretin receptor and for a secretin receptor spliceoform different from those occurring in lung and gut tumors. In conclusion, secretin receptors are new markers for bronchopulmonary carcinoid tumors, and represent the molecular basis for an in vivo targeting of carcinoid tumors for diagnosis and therapy. Furthermore, secretin receptors may play a role in peritumoral lung pathophysiology. Secretin receptor mis-splicing specifically occurs in tumor and non-tumor lung pathology.
Assuntos
Biomarcadores Tumorais/análise , Tumor Carcinoide/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Autorradiografia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Expressão Gênica , Humanos , Pulmão/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.
Assuntos
Proliferação de Células , Óxido de Deutério/análise , Animais , Água Corporal , Morte Celular , Colo/citologia , Replicação do DNA , Óxido de Deutério/metabolismo , Células Endoteliais/citologia , Células Epiteliais/citologia , Humanos , Marcação por Isótopo , Queratinócitos/citologia , Camundongos , Células Musculares/citologia , Neoplasias , Neurônios/citologia , Ratos , Técnicas de Cultura de TecidosRESUMO
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.
Assuntos
Proliferação de Células , Cromatografia Gasosa/métodos , DNA/metabolismo , Óxido de Deutério/análise , Espectrometria de Massas/métodos , Animais , DNA/biossíntese , Replicação do DNA , Desoxirribose/análogos & derivados , Desoxirribose/química , Desoxirribose/metabolismo , Óxido de Deutério/metabolismo , Humanos , Marcação por Isótopo , Conformação MolecularRESUMO
BACKGROUND & AIMS: Pancreatic and bile duct carcinomas represent highly aggressive malignancies that evolve from secretin receptor-rich ductular cells. With premessenger RNA splicing abnormalities common in cancer, we evaluated whether an abnormal secretin receptor spliceoform were present, characterized it, and developed a serum assay for it. METHODS: Cancer cell lines and healthy and neoplastic tissue were studied by nested reverse-transcription polymerase chain reaction and sequencing. A promising spliceoform was isolated and characterized, and monoclonal antibodies were raised to 2 distinct regions. A dual antibody enzyme-linked immunosorbent assay was developed and applied to blinded serum samples from 26 patients with pancreatic carcinoma, 10 patients with chronic pancreatitis, and 14 controls. RESULTS: Each of 9 pancreatic cancer specimens and no normal tissue expressed a secretin receptor variant with exons 3 and 4 deleted. This encoded a 111-residue peptide with its first 43 residues identical to wild-type receptor, but, subsequent to a shift in coding frame and early truncation, the next 68 residues were unique in the transcriptome/proteome. This nonfunctional soluble protein did not bind or signal in response to secretin and was secreted from transfected MiaPaCa-2 cells. Elevated serum levels of this variant were present in 69% of pancreatic cancer patients, 60% of chronic pancreatitis patients, and 1 of 14 controls. CONCLUSIONS: We identified a novel abnormal spliceoform of the secretin receptor in pancreatic and bile duct cancers and developed a dual antibody sandwich enzyme-linked immunosorbent assay to measure it in the circulation. Initial application of this assay in patients with pancreatic cancer and chronic pancreatitis was promising, but additional validation will be required to evaluate its clinical utility.
Assuntos
Colangiocarcinoma/diagnóstico , Colangiocarcinoma/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Biomarcadores/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Dados de Sequência Molecular , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/imunologiaRESUMO
BACKGROUND/AIMS: Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer. METHODS: Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using (125)I-[Tyr(10)] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR. RESULTS: Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas. CONCLUSIONS: The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.
Assuntos
Neoplasias dos Ductos Biliares/genética , Ductos Biliares Extra-Hepáticos/metabolismo , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , Animais , Autorradiografia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Extra-Hepáticos/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Mensageiro/biossíntese , Ratos , Receptores Acoplados a Proteínas G/biossíntese , Receptores dos Hormônios Gastrointestinais/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SecretinaRESUMO
Receptors for gut hormones, which are often overexpressed in cancer, are clinically relevant for receptor-targeted tumor imaging and therapy. Because the receptors for the gut hormone secretin are poorly characterized, we assessed secretin receptor expression in the main secretin target, the human pancreas. We investigated 58 non-neoplastic pancreases and 55 pancreatic tumors for receptor localization and density by in vitro receptor autoradiography using [(125)I]Tyr(10) rat secretin and for secretin receptor mRNA by reverse transcriptase-polymerase chain reaction. Secretin receptors were highly expressed in non-neoplastic ducts and lobuli and also in lower amounts in ductal neoplasias, including ductal adenocarcinoma, intraductal papillary mucinous tumors, and pancreatic intraepithelial neoplasia. Reverse transcriptase-polymerase chain reaction revealed wild-type receptor mRNA in the non-neoplastic pancreas and both wild-type and spliced variant receptor transcripts in ductal adenocarcinomas. Serous cystic tumors were highly positive for secretin receptors, whereas mucinous cystic tumors were negative. This study is the first to describe the precise secretin receptor distribution in human non-neoplastic pancreas and various pancreatic tumors. High secretin receptor expression in the non-neoplastic ducts reflects the major role of secretin in bicarbonate secretion. Reduced secretin binding in pancreatic ductal tumors may relate to (alternatively spliced) secretin receptor isoforms. Thus, secretin receptors in pancreatic tumors may represent potential clinical targets.