RESUMO
We report here the complete genome sequence of a WU polyomavirus (WUPyV) isolate, also known as human polyomavirus 4, collected in 2016 from a patient in Arkansas with an acute respiratory infection. Isolate hPyV4/USA/AR001/2016 has a double-stranded DNA genome of 5,229 bp in length.
RESUMO
We have recently reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits epidermal growth factor (EGF) withdrawal-induced apoptosis in the human mammary epithelial cell line MCF-10A. We hypothesized that TCDD-mediated inhibition of apoptosis was due to its ability to stimulate the EGF receptor (EGFR) pathway. Indeed, in the present studies, the EGFR inhibitor AG1478 was able to prevent TCDD-, EGF-, and transforming growth factor alpha (TGF-alpha)-dependent cell recovery and inhibition of apoptosis. These effects were specific for an EGFR-mediated pathway because cotreatment with AG825, an erbB2 inhibitor, had little effect on apoptosis. In addition, TCDD was able to mimic the EGF and TGF-alpha signaling as demonstrated by increasing Akt and extracellular signal-regulated kinase 1,2 phosphorylation. These effects were dependent on EGFR activity because AG1478, but not AG825, was able to prevent EGF-, TGF-alpha, or TCDD-mediated Akt and extracellular signal-regulated kinase 1,2 phosphorylation. The ability of TCDD to stimulate the EGFR pathway and inhibit apoptosis may be due to the ability of TCDD to increase expression of TGF-alpha, a ligand for EGFR. Treatment with 10 nM TCDD increased TGF-alpha mRNA at 2 h and TGF-alpha protein at 6 h. These data suggest a mechanism whereby TCDD is able to inhibit apoptosis in human mammary epithelial cells by stimulating TGF-alpha production, resulting in an autocrine effect.
Assuntos
Apoptose/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Proteínas Serina-Treonina Quinases , Fator de Crescimento Transformador alfa/biossíntese , Apoptose/fisiologia , Benzotiazóis , Mama/citologia , Mama/efeitos dos fármacos , Mama/metabolismo , Contagem de Células , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Quinazolinas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/genética , Tirfostinas/farmacologiaRESUMO
With the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry, human melanoma cell lines (Colo 38, M-16, and M-21) were examined for the quantitative level of expression of HLA-A,B antigens, Ia-like antigens, and two human melanoma-associated antigens (MAA) referred to as 280K and 94K MAAs. Each of the melanoma cell lines examined showed tremendous heterogeneity with regard to antigen expression. A detailed study of the heterogeneity of the four antigens listed revealed that variation in cell size could, in part, account for the large differences in antigen expression observed. Cell surface density of HLA-A,B antigens, Ia-like antigens, and the 280K and 94K MAAs remained relatively constant over a wide range of cell sizes that were examined, with the exception that small melanoma cells showed a slightly lower mean surface density of MAA expression than did large cells. A novel method was used to detect the expression of the two MAAs as a function of the Colo 38 cell cycle using dual-laser beam flow cytometry. The results of these experiments show that both the 280K MAA and the 94K MAA are differentially expressed during various stages of the cell cycle, with each antigen being maximally detected during G2 + M.
Assuntos
Anticorpos Monoclonais , Antígenos de Neoplasias/análise , Antígenos HLA/análise , Antígenos de Histocompatibilidade Classe II/análise , Melanoma/imunologia , Proteínas de Neoplasias/análise , Linhagem Celular , Membrana Celular/imunologia , Citometria de Fluxo , Imunofluorescência , Antígenos HLA-A , Antígenos HLA-B , Humanos , Leucemia Linfoide/imunologia , Antígenos Específicos de MelanomaRESUMO
Using target capture of viral nucleic acid and next-generation sequencing, we generated the complete genomes of two novel human parainfluenza virus 1 isolates. Isolates AR001 (accession no. KX570602) and NM001 (accession no. KX639498) were collected 3 months apart from pediatric patients with acute respiratory infection from Arkansas and New Mexico, respectively.
RESUMO
Techniques for the rapid and efficient purification of mouse monoclonal antibodies from murine ascites are described that utilize anion exchange and gel permeation chromatography using high performance liquid chromatography (HPLC). Anion exchange chromatography was performed at neutral pH using a hydrophilic resin conjugated with a substituted amine (Mono Q column, Pharmacia Fine Chemicals). Various subclasses of mouse IgG monoclonals were assessed for their binding to this matrix, with all of the antibodies tested eluting at relatively low concentrations of sodium chloride. In some instances, a protein tentatively identified as transferrin was co-purified using this anion exchange procedure. However, this protein was easily removed from the IgG using gel permeation chromatography (Bio-Sil TSK-250, Bio-Rad), also performed at neutral pH.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Líquido Ascítico/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Cabras , Humanos , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Camundongos , Coelhos , OvinosRESUMO
These studies were conducted to determine the biodistribution and pharmacokinetics of [99mTc]metallothionein-conjugated B72.3 ([ Tc]MT-B72.3) in Rhesus monkeys (Macaca mulatta) that were performed as part of the preclinical evaluation of [Tc]MT-B72.3. The B72.3-MT conjugate was studied at three doses of B72.3 ranging from 0.03 mg/kg to 1 mg/kg to determine whether a relationship existed between the dose of total antibody administered intravenously and the biodistribution and clearance of the radiolabeled protein. Results indicated that [Tc]MT-B72.3 distributes rapidly to central body cavity organs and that there was no difference in the rate of blood elimination for the three doses of B72.3 studied. The terminal phase of blood elimination was found to be 26.2 +/- 6.1 hr for the combined groups of monkeys. Approximately one-half of injected 99mTc activity was recovered in the urine within 24 hr. A second purpose of these studies was to evaluate the overall immunogenicity of the mouse monoclonal B72.3 IgG1 antibody in Rhesus monkeys. These results demonstrated that a single i.v. exposure to mouse monoclonal B72.3 at doses of 0.3 mg/kg or greater elicited antibody production to B72.3 in Rhesus monkeys within 3 wk. Analysis of [Tc]MT-B72.3 biodistribution and clearance in monkeys with circulating levels of antibodies to B72.3 (immunized monkeys) revealed that the liver was the primary site of clearance of the presumed immune complex and that blood elimination was greatly accelerated.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Metalotioneína , Compostos Organometálicos , Compostos de Organotecnécio , Tecnécio , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Injeções Intravenosas , Macaca mulatta , CamundongosRESUMO
The article highlighted in this issue is "An Aryl Hydrocarbon Receptor Independent Mechanism of JP-8 Jet Fuel Immunotoxicity in Ah-Responsive and Ah-Nonresponsive Mice" by Andrew C. Dudley, Margie M. Peden-Adams, Jackie EuDaly, Richard S. Pollenz, and Deborah E. Keil (pp. 251-259).
Assuntos
Genômica , Proteoma/análise , Toxicologia/métodos , Animais , DNA/análise , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/genética , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
A novel and successful undergraduate-level course in pharmacology has been established as part of the drug education program at California's San Quentin State Prison. The program was designed to create a two-way teaching--learning experience between former drug abusers and their teachers, all of whom were doctoral candidates and post-doctoral scholars in pharmacology at a nearby university medical center. In reviewing the pharmacology of psychoactive drugs, the emphasis was on the principles of drug action in the central nervous system, with additional attention to those factors that contribute a potential for abuse. An attempt was made to present information in a strictly objective and non-prejudical manner. An initial analysis has shown a possible change in attitude among the student-inmates toward drugs of abuse. It is likely that courses following a similar design would be well received and successful in any type of undergraduate program that addresses the subject of drug abuse.
Assuntos
Farmacologia/educação , Prisioneiros , Transtornos Relacionados ao Uso de Substâncias/educação , Adulto , Atitude , California , Currículo , Estudos de Avaliação como Assunto , Docentes , Humanos , Masculino , Materiais de EnsinoRESUMO
Polycyclic aromatic hydrocarbons affect cells in many ways, including covalent modifications of DNA, participation in redox cycling, and alterations in cellular signaling pathways. Similarly, exposure to ultraviolet (UV) light may modify DNA, generate reactive oxygen species, and alter signaling. Because environmental conditions may interact to affect cellular functions, we investigated the combined effects of benzo[a]pyrene (BaP) and UV light in a cell line in which BaP-induced alterations in Ca(2+) homeostasis have previously been shown. Exposure of MCF-10A cells to BaP (18 h) followed by a brief (5 min) exposure to UVA resulted in resistance to trypsinization of cells grown on type I collagen (Vitrogen). This effect was not seen following treatment with BaP or UVA alone nor with benzo(e)pyrene (BeP)+UVA. BaP+UVA light also caused actin filaments to reorganize from typical stress fibers to substrate-associated aggregates of actin and caused depletion of cellular adenosine triphosphate (ATP). The effects of BaP+UVA on adhesion and actin aggregate formation were partially prevented by treatment with reduced glutathione. Depletion of cellular ATP affected resistance to trypsinization and actin organization in a similar manner. Thus, these studies suggest a redox-sensitive interaction between BaP+UVA light to deplete cellular ATP levels, resulting in resistance to trypsinization and actin filament reorganization in MCF-10A cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Benzo(a)pireno/farmacologia , Citoesqueleto/efeitos dos fármacos , Tripsina/farmacologia , Raios Ultravioleta , Actinas/efeitos dos fármacos , Actinas/metabolismo , Actinas/efeitos da radiação , Antimicina A/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Contagem de Células , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Ácido Egtázico/farmacologia , Glutationa/farmacologia , Humanos , Estresse OxidativoRESUMO
Flow cytometry Coulter volume analysis was used to examine the effects of an acute exposure to cadmium or lead on subpopulations of Balb/c bone marrow cells. A significant shift in the volume of Balb/c bone marrow cells was detected in response to a single i.p. injection of cadmium acetate (Cd) or lead acetate (Pb) compared to sodium acetate (Na)-treated mice. An increase in the relative number or size of myeloid/monocytic cells was noted in the bone marrow of cadmium or lead-treated mice. This effect was more pronounced in aged Balb/c mice than in young adults. These studies suggest the flow cytometry Coulter volume analysis may be a useful and sensitive technique for the assessment of cellular changes that occur in the bone marrow in response to xenobiotic exposure.
Assuntos
Medula Óssea/efeitos dos fármacos , Intoxicação por Cádmio/patologia , Intoxicação por Chumbo/patologia , Envelhecimento , Animais , Células da Medula Óssea , Contagem de Células , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
It has been well established that certain polycyclic aromatic hydrocarbons (PAHs), such as 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3MC), and benzo[a]pyrene (BaP), produce immunotoxicity and cancer in rodents and that these effects are also likely seen in humans. Our laboratory has found that polycyclic aromatic hydrocarbons (PAHs) produce an increase in intracellular Ca2+ in lymphocytes that appears to correlate with their immunotoxicity. Specifically, immunotoxic PAHs, such as DMBA and BaP, have been shown to produce a sustained increase in intracellular Ca2+ in lymphocytes, whereas nonimmunosuppressive PAHs, such as benzo[e]pyrene (BeP) and anthracene, do not. Our studies previously demonstrated that the rapid increase in intracellular Ca2+ produced by DMBA in HPB-ALL T cells was caused by protein tyrosine kinase (PTK) activation in human T cells, leading to tyrosine phosphorylation of phospholipase C (PLCgamma) and IP3-dependent Ca2+ mobilization. However, the specificity of PTK activation by PAHs was not established. In the present studies, we extend our observations of PTK activation by examining a number of PAHs for their effects on total and specific (Fyn and ZAP-70) PTK activity. We show that 10 microM concentrations of PAHs nonspecifically and rapidly (within 5 min) stimulate PTKs in the HPB-ALL human T cell line. BeP and anthracene were found to be nearly as effective at increasing total tyrosine kinase activity as DMBA, 3MC, and BaP, observed 5 min after exposure. We found that only immunotoxic PAHs activated the Fyn and ZAP-70 PTKs at 10 min, but total PTK activity was still increased by nonimmunotoxic PAHs, BeP, or anthracene after 10 min of exposure. These studies demonstrate that immunotoxic PAHs increase total and specific PTK activity in the human HPB-ALL T cell line. Thus the rapid increase in PTK activity produced by PAHs may not correlate with the immunotoxicity of these agents.
Assuntos
Carcinógenos Ambientais/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Análise de Variância , Antracenos/farmacologia , Benzo(a)pireno/farmacologia , Benzopirenos/farmacologia , Linhagem Celular , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos/enzimologia , Relação Estrutura-AtividadeRESUMO
A case report is presented describing a 27-year-old woman with invasive trophoblastic hydatidiform mole metastatic to the lung. Gamma scintiscanning, using a polyclonal and monoclonal antibody specific to human chorionic gonadotropin, hCG, and labeled with Tc-99m, is described. The area of the primary lesion in the uterus was demonstrated with both antibodies tested without computer subtraction techniques; metastatic deposits in the lung were detected only with the aid of blood pool subtraction techniques.
Assuntos
Anticorpos , Gonadotropina Coriônica/imunologia , Mola Hidatiforme/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Tecnécio , Neoplasias Uterinas/diagnóstico por imagem , Adulto , Anticorpos/administração & dosagem , Anticorpos/imunologia , Feminino , Humanos , Neoplasias Pulmonares/secundário , Gravidez , Cintilografia , Técnica de SubtraçãoAssuntos
Anticorpos Monoclonais/isolamento & purificação , Animais , Linhagem Celular , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/instrumentação , Cromatografia por Troca Iônica/métodos , Imunoglobulina G/isolamento & purificação , Camundongos , Espectrofotometria UltravioletaRESUMO
The potential health effects of inhaling carbon nanotubes are important because of possible exposures in occupational settings. Previously, we have shown mice that have inhaled multiwalled carbon nanotubes have suppressed systemic immune function. Here, we show the mechanisms for this immune suppression. Mice were exposed to 0, 0.3 or 1 mg m(-3) multiwalled carbon nanotubes for 6 h per day for 14 consecutive days in whole-body inhalation chambers. Only those exposed to a dose of 1 mg m(-3) presented suppressed immune function; this involved activation of cyclooxygenase enzymes in the spleen in response to a signal from the lungs. Spleen cells from exposed animals partially recovered their immune function when treated with ibuprofen, a drug that blocks the formation of cyclooxygenase enzymes. Knockout mice without cyclooxygenase enzymes were not affected when exposed to multiwalled carbon nanotubes, further confirming the importance of this enzyme in suppression. Proteins from the lungs of exposed mice suppressed the immune function of spleen cells from normal mice, but not those from knockout mice. Our findings suggest that signals from the lung can activate signals in the spleen to suppress the immune function of exposed mice.
Assuntos
Sistema Imunitário/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/deficiência , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Modelos Animais de Doenças , Ibuprofeno/farmacologia , Exposição por Inalação/efeitos adversos , Pulmão/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho da Partícula , Baço/citologia , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The effects of PGI2, PGD2, and PGF2 alpha on human peripheral blood lymphocytes (HPBL) cyclic AMP levels and their response to a T cell mitogen (PHA) were investigated. It was determined that although PGI2, PGD2, and PGE2 all elevate intracellular levels of cyclic AMP in HPBL, only PGD2 and PGE2 exert potent suppression of PHA-induced lymphocyte proliferation. It is suggested that the instability of PGI2 in aqueous solution is at least partially responsible for its relative inability to maintain elevated levels of cyclic AMP and concomitant suppression of mitogenesis. PGD2 was found to exert an effect very similar to PGE2, both in terms of cyclic AMP elevation and suppression of T cell mitogenesis.
Assuntos
AMP Cíclico/farmacologia , Epoprostenol/farmacologia , Prostaglandinas D/farmacologia , Prostaglandinas/farmacologia , Linfócitos T/imunologia , Adulto , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Prostaglandinas E/farmacologia , Prostaglandinas F/farmacologiaRESUMO
Cyclic AMP-elevating agents have been shown to increase the expression of Fc receptors on the Abelson virus-induced pre-B cell tumor ABE-8. Such agents included isoproterenol, PGE1, 3-isobutyl-1-methyl xanthine, and 8 BrcAMP. The positive inductive effect produced by these agents was inhibited by 8 BrcGMP and PGF2 alpha, which putatively elevate cyclic GMP levels. Other agents also shown to induce Fc receptor expression were LPS and certain batches of fetal calf sera. In contrast to the inductive effect produced by cyclic AMP elevating agents, 8 BrcGMP and PGF2 alpha were unable to reverse the increased Fc receptor expression produced by LPS and fetal calf sera. Thus, these latter agents may act via a qualitatively different mechanism in producing a change in phenotypic expression. The results of this study are discussed in terms of the use of tumor cells as a model system for studying the pharmacologic control of lymphocyte differentiation.
Assuntos
Linfócitos B/imunologia , AMP Cíclico/farmacologia , Linfoma/imunologia , Receptores Fc/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular , Membrana Celular/imunologia , Células Cultivadas , GMP Cíclico/farmacologia , Relação Dose-Resposta Imunológica , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , OvinosRESUMO
During the past decade there has been significant progress made in understanding how environmental agents, drugs, certain chemicals present in the diet, and occupational agents affect the immune system of animals and humans. Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmentally prevalent xenobiotics that exert complex effects on the immune system. These agents, typified by benzo(a)pyrene (BaP), have been shown to alter antigen and mitogen receptor signaling pathways, leading to suppression of humoral and cell-mediated immunity, and at high exposure levels to activation of genes involved in apoptosis in lymphoid cells. Interestingly, at low exposure levels, PAHs may actually augment cell signaling pathways, resulting in immune enhancement or an adjuvant effect. While the biochemical targets and mechanisms responsible for immune modulation are still under investigation, several themes are evolving. PAHs, principally through their cytochrome-P450-derived metabolites, activate oxidative and electrophilic signaling pathways in lymphoid and nonlymphoid cells, including myeloid, epithelial, and other cells. Although PAHs affect signaling pathways in nonlymphoid cells leading to complex interactions between antigen-specific and nonspecific immune and inflammatory responses, this brief review focuses on the mechanisms of signaling by environmentally prevalent PAHs in human lymphocytes. Understanding the mechanisms by which xenobiotics alter adaptive and nonadaptive immune responses may shed light on the etiology of environmental and occupational immune diseases.
Assuntos
Poluentes Ambientais/análise , Linfócitos/imunologia , Hidrocarbonetos Policíclicos Aromáticos/análise , Animais , Poluentes Ambientais/toxicidade , Humanos , Linfócitos/citologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Transdução de SinaisRESUMO
The purpose of the experiments described in these studies was to determine the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on B-cell activation produced by anti-IgD antibodies and interleukin-4 (IL-4). B and T cells are known to share many of the same biochemical pathways for cell activation by mitogen and antigen receptors. Previous studies in this laboratory have shown that DMBA inhibits mitogen-induced Ca2+ mobilization in murine and human T cells and produces an increase in intracellular Ca2+ in resting cells. The results of the present studies demonstrate that DMBA increases Ca2+ in resting B cells and inhibits B cell activation produced by anti-IgD antibodies, as measured by mobilization of free intracellular Ca2+ and [3H]thymidine incorporation. The proliferative response of B cells to insolubilized anti-IgD was suppressed only when cells were preexposed to DMBA. In contrast, IL-4 pathways of B-cell activation were insensitive to inhibition by DMBA, even when cells were preexposed. The induction of Class II MHC antigen (Ia) antigens on B cells by IL-4 was also found to be insensitive to DMBA treatment. These results suggest that DMBA suppresses only Ca(2+)-dependent pathways of B cell activation and indicate that altered Ca2+ homeostasis may be responsible for immunosuppression induced by this agent.
Assuntos
9,10-Dimetil-1,2-benzantraceno/farmacologia , Linfócitos B/efeitos dos fármacos , Cálcio/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Cálcio/metabolismo , Cálcio/fisiologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/fisiologia , Imunoglobulina D/imunologia , Interleucina-4/farmacologia , Líquido Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Baço/citologia , Baço/efeitos dos fármacos , Estimulação QuímicaRESUMO
Numerous studies have demonstrated an association between polycyclic aromatic hydrocarbons (PAHs) and lymphocyte toxicity. The present study shows that, consistent with its effects on Ca2+ homeostasis, benzo[a]pyrene (BaP) induces apoptosis in Daudi cells. Terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis at 18 h revealed a significant increase in the number of cells undergoing apoptosis in response to BaP (75%), BaP-7, 8-dihydrodiol (110%), and BaP-7,8-9,10-diol epoxide (BPDE) (215%) over DMSO vehicle control cultures. By 36 h, the trend toward increasing numbers of apoptotic cells continued with the parent compound producing a 125% increase over control values and the 7, 8-dihydrodiol and BPDE metabolites producing 195% and 370% increases over controls, respectively. DNA fragmentation assays demonstrated the presence of internucleosomal cleavage products consistent with the increasing numbers of TUNEL-positive cells responding to PAHs at 18 and 36 h. Analysis of poly(ADP-ribose) polymerase (PARP) protein in BaP- and BaP-7,8-dihydrodiol-treated cells strongly suggested the involvement of cysteine proteases by the appearance of an 85-kD fragment derived from hydrolytic cleavage of PARP, a phenomenon that has been associated with apoptosis in many systems. Immunoblot analysis demonstrated that both BaP and its 7,8-dihydrodiol metabolite affected a pathway involving Bcl-2 and Bax cytosolic proteins. Daudi cells undergoing apoptosis at 36 h in response to 10 microM BaP, the parent compound, expressed moderately reduced amounts of Bcl-2 (78% of vehicle controls). At the same time point, the 7,8-dihydrodiol and BDPE metabolites at 3 microM resulted in Bcl-2 protein expression that was 52% of that seen in vehicle controls. Parallel samples analyzed for expression of Bax protein displayed a 130% increase over vehicle control in Bax expression in response to the parent compound, while the 7,8-dihydrodiol metabolite produced a 257% increase in Bax. Furthermore, the effects on increased Bax expression were observed as early as 3 h after PAH exposure. The apoptotic response to PAHs in Daudi cells was sensitive to 4-h pretreatment with 0.3 microM alpha-naphthoflavone (ANF), a known inhibitor of cytochrome P450. In TUNEL assays of cells exposed to PAHs following pretreatment with ANF, at 18 h there was a significant reduction in the number of cells undergoing apoptosis in response to ANF compared to cells that were not pretreated with the compound. The effect of the parent compound at 18 h was completely blocked with ANF pretreatment, while ANF exerted a relatively weaker, but significant, effect on BaP-7, 8-dihydrodiol-induced apoptosis. With regard to modulation of expression of apoptosis-related proteins, Bax expression was restored to that observed in vehicle-control cultures at all time points tested (3, 18, and 36 h). Bcl-2 expression was most responsive to ANF at later time points following PAH exposure (18 and 36 h); however, Bcl-2 appeared to be more sensitive to the effects of ANF alone. Taken together, these data suggest that modulation of Bcl-2 family proteins, perhaps secondary to altered Ca2+ homeostasis, plays an important role in human B cell apoptosis induced by BaP.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Benzo(a)pireno/toxicidade , Di-Hidroxi-Di-Hidrobenzopirenos/toxicidade , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/farmacologia , Fator Natriurético Atrial/farmacologia , Linhagem Celular , Fragmentação do DNA , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2RESUMO
We have compared the stimulatory activity of DBcAMP with various antiproliferative agents on the induction of the humoral immune response. When they are present only during an early stage of immune induction, DBcAMP, colcemid, cytosine-arabinoside, hydroxy urea, and high specific activity 3H-thymidine can all enhance the primary 19s antibody response to SRBC. In contrast, each of these agents inhibits the PFC response, when they are incubated with the cells during late stages of induction of humoral immunity. Because all of these agents can inhibit proliferation of cultured cells, the results suggested that DBcAMP and other agents that elevate cAMP could augment humoral immunity via their effects on cellular proliferation. However, we also found that although each agent could modulate induction of the immune response to SRBC, only DBcAMP produced a dose- and time-dependent augmentation of the response to DLF. We conclude that although antiproliferative effects of drugs may contribute to augmentation of some humoral antibody responses, this effect alone is insufficient to account for the mechanism by which agents that elevate intracellular levels of cAMP produce enhancement of humoral immunity.