RESUMO
BACKGROUND: Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a rare disease which can cause severe morbidity and mortality. The aim of this study is to evaluate the clinical manifestation and course of DRESS syndrome. METHODS: We conducted a retrospective analysis of prospectively collected data in 45 patients with DRESS syndrome diagnosed between September 2009 and August 2011. RESULTS: The most common causative drug group was antibiotics (n=13, 28.9%), followed by anticonvulsants (n=12, 26.7%), antituberculosis drugs (n=6, 13.3%), non-steroidal anti-inflammatory drugs (n=4, 8.9%), undetermined agents (n=4, 8.9%), allopurinol (n=3, 6.7%), and others (n=3, 6.7%). The latency period ranged from 2 to 120 days, with a mean of 20.2 ± 24.3 days. The longest latency period was noted for the antituberculosis drug group, at 46.5 ± 29.9 days. Eosinophilia in peripheral blood examination was noted in 35 subjects (77.8%). Atypical lymphocytosis was noted in 16 patients (35.6%), and thrombocytopenia in seven patients (15.6%). Hepatic involvement was noted in 39 (86.7%) study patients, kidney in eight (17.8%), lung in four (8.9%), and central nervous system in one (2.3%). Systemic corticosteroids were administered to 10 patients (22.2%). Forty-three patients (95.6%) showed complete recovery, while two patients had poor outcomes. CONCLUSIONS: DRESS syndrome was not more uncommon than generally recognised. Antibiotics were the most frequently implicated drug group, followed by anticonvulsants. Most patients with this disease showed a better clinical outcome than that which had been generally expected.
Assuntos
Corticosteroides/administração & dosagem , Síndrome de Hipersensibilidade a Medicamentos/diagnóstico , Eosinofilia/diagnóstico , Rim/patologia , Fígado/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/imunologia , Antibacterianos/imunologia , Anticonvulsivantes/imunologia , Antituberculosos/imunologia , Síndrome de Hipersensibilidade a Medicamentos/tratamento farmacológico , Síndrome de Hipersensibilidade a Medicamentos/imunologia , Eosinofilia/tratamento farmacológico , Eosinofilia/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: The prevalence of allergic bronchopulmonary aspergillosis (ABPA) in patients with bronchial asthma remains unknown. We evaluated the roles of various laboratory tests in the diagnosis of ABPA, including, skin prick test (SPT) for Aspergillus fumigatus (Af), and serum Af specific IgE and IgG antibody measurement. METHODS: A total of 50 asthma patients with more than 1000cell/µL of peripheral blood eosinophils were prospectively collected between January 2007 and September 2011. Evaluations using SPT for Af, serum total IgE and specific IgE antibody to Af by CAP system, IgG antibody to Af by enzyme immunoassay (EIA) or CAP system were performed according to the essential minimal criteria for the diagnosis of ABPA - asthma, immediate cutaneous reactivity to Af, elevated total IgE, and raised Af specific IgE and IgG. RESULTS: Among 50 patients, three patients (6.0%) were diagnosed as ABPA, of whom each confirmed five items of the essential minimal diagnostic criteria for the diagnosis of ABPA. Six patients (12.0%) showed negative responses to Af in SPT, but positive responses in specific IgE by CAP system. Eight patients (16.0%) showed negative responses to IgG to Af by CAP system, but positive responses by enzyme immunoassay (EIA). CONCLUSIONS: SPT and serum IgE to Af measurement by CAP system should be performed simultaneously. It is reasonable to set up cut-off values in Af specific IgE/IgG by CAP system for the differentiation of ABPA from Af sensitised asthma patients.
Assuntos
Aspergilose Broncopulmonar Alérgica/complicações , Aspergilose Broncopulmonar Alérgica/diagnóstico , Aspergilose Broncopulmonar Alérgica/epidemiologia , Asma/complicações , Adulto , Idoso , Anticorpos Antifúngicos/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Prevalência , Testes Cutâneos , Adulto JovemRESUMO
Indole-3-carbinol (I3C), a natural product of Brassica vegetables such as broccoli and cabbage, inhibits proliferation and induces apoptosis in various cancer cells. I3C has recently received attention as a possible anti-obesity agent. However, how I3C interacts with specific targets in the pathways involved in obesity and metabolic disorders is unknown. Silent mating type information regulation 2 homolog 1 (SIRT1), a NADþ-dependent deacetylase sirtuin, has recently emerged as a novel therapeutic target for metabolic diseases. Herein, we report that I3C is a potent, specific SIRT1 activator efficacious in cultured 3T3-L1 cell lines. A pull-down assay showed that I3C binds to SIRT1. To assess the significance of this binding, we determined whether I3C could activate SIRT1 deacetylase activity in a cell-free system. We found that I3C binds to SIRT1 and activates SIRT1 deacetylase activity in 3T3-L1 cells. In addition, I3C did not inhibit adipocyte differentiation in 3T3-L1 cells in which SIRT1 was knockdowned. Further, reverse transcriptase polymerase chain reaction analysis showed that I3C treatment reduced mRNA levels of adipogenic genes that encode for C/EBPa, PPARg2, FAS, and aP2 in 3T3-L1 cells but not in SIRT1 knockdown cells. Overall, these results suggested that I3C ameliorates adipogenesis by activating SIRT1 in 3T3-L1 cells.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Indóis/farmacologia , Obesidade/metabolismo , Sirtuína 1/efeitos dos fármacos , Células 3T3-L1/efeitos dos fármacos , Células 3T3-L1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
BACKGROUND: The clinical features of drug-induced hypersensitivity syndrome (DIHS) or drug rash with eosinophilia and systemic symptoms (DRESS) syndrome are complicated, and the incidence of this condition is very low. OBJECTIVE: To evaluate the clinical course of DIHS/DRESS and identify effective treatment options. METHODS: This study was a retrospective analysis of prospectively collected clinical data in 38 consecutive patients with DIHS/DRESS diagnosed between March 2004 and January 2009. We investigated the clinical features, response to treatment, and outcome of 38 patients. RESULTS: The study patients consisted of 18 men (47.4%) and 20 women (52.6%). The most common causative drugs were anticonvulsants (47.4%) and antibiotics (18.4%), followed by nonsteroidal anti-inflammatory drugs (NSAIDs) (13.2%), allopurinol (5.3%), and undetermined agents (15.8%). The latency period ranged from 3 to 105 days, with a mean (SD) of 25.2 (21.5) days. Systemic corticosteroids were administered to 16 patients (42.1%). Twenty-two (57.9%) patients were treated with topical corticosteroids and antihistamines (no systemic corticosteroids). Complete recovery was noted in 36 patients (94.8%). Two of the patients treated with systemic corticosteroids had a poor outcome: one died due to an opportunistic infection secondary to long-term systemic corticosteroid treatment; the other showed progressive deterioration of liver damage, although the final outcome is not known. CONCLUSION: The drugs associated with DIHS/DRESS were variable and most frequently included anticonvulsants, beta-lactam antibiotics, and NSAIDs. The syndrome was more common than generally recognized. Additional studies are needed to evaluate the clinical indications for systemic corticosteroids in patients with DIHS/DRESS.
Assuntos
Hipersensibilidade a Drogas/etiologia , Administração Oral , Administração Tópica , Corticosteroides/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Anti-Inflamatórios não Esteroides/efeitos adversos , Anticonvulsivantes/efeitos adversos , Hipersensibilidade a Drogas/tratamento farmacológico , Hipersensibilidade a Drogas/imunologia , Feminino , Antagonistas dos Receptores Histamínicos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estatísticas não Paramétricas , Adulto JovemRESUMO
Hypersensitivity pneumonitis (HP) can be caused by drugs administered via routes other than the airway. We report a case of HP caused by intravesical bacille Calmette-Guerin (BCG) administered for the treatment of bladder cancer. We attempt to identify the causative agents of HP. A 60-year-old, nonsmoking homemaker was referred to our hospital with nonresolving pneumonia. The patient had dyspnea, cough, and fever that started after 3 weekly cycles of intravesical BCG. High-resolution computed tomography of the chest revealed multiple tiny nodules and ground-glass opacities on both lung fields. Pulmonary function tests revealed a restrictive ventilatory defect with decreased diffusion capacity. Histopathology of the transbronchial lung biopsy specimens showed immature noncaseating granulomata. Immunoblotting analysis of serum and BCG demonstrated more than 10 immunoglobulin G fractions binding to BCG. This case illustrates that HP can be caused by intravesical instillation of BCG.
Assuntos
Alveolite Alérgica Extrínseca/diagnóstico , Alveolite Alérgica Extrínseca/imunologia , Imunoterapia/efeitos adversos , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/terapia , Administração Intravesical , Alveolite Alérgica Extrínseca/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias da Bexiga Urinária/imunologiaRESUMO
Retinoic acid (RA) has broad clinical applications for the treatment of various cancers, particularly acute promyelocytic leukemia. However, RA-based therapy is limited by relapse in patients associated with RA resistance, the mechanism of which is poorly understood. Here, we suggest a new molecular mechanism of RA resistance by a repressor, named RA resistance factor (RaRF). RaRF suppressed transcriptional activity of the RA receptor (RAR) by directly interacting with and sequestering RAR to the nucleolus in response to RA. RaRF was highly expressed in RA-resistant leukemia cells and its expression was strongly correlated with RA sensitivity. MCL1 was upregulated by RA treatment upon RaRF depletion, accompanying leukemic myeloblast differentiation, which is negatively regulated by ectopic RaRF expression. Collectively, we propose that RaRF may be a factor in the resistance mechanism and thus a potential target for leukemia therapy using RA.
Assuntos
Nucléolo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Promielocítica Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Tretinoína/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Regulação Leucêmica da Expressão Gênica , Células Precursoras de Granulócitos/efeitos dos fármacos , Células Precursoras de Granulócitos/patologia , Humanos , Estimativa de Kaplan-Meier , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/mortalidade , Leucemia Promielocítica Aguda/patologia , Receptores do Ácido Retinoico/genética , Regulação para CimaRESUMO
Estrogen receptor alpha (ERα) has a pivotal role in breast carcinogenesis by associating with various cellular factors. Selective expression of additional sex comb-like 2 (ASXL2) in ERα-positive breast cancer cells prompted us to investigate its role in chromatin modification required for ERα activation and breast carcinogenesis. Here, we observed that ASXL2 interacts with ligand E2-bound ERα and mediates ERα activation. Chromatin immunoprecipitation-sequencing analysis supports a positive role of ASXL2 at ERα target gene promoters. ASXL2 forms a complex with histone methylation modifiers including LSD1, UTX and MLL2, which all are recruited to the E2-responsive genes via ASXL2 and regulate methylations at histone H3 lysine 4, 9 and 27. The preferential binding of the PHD finger of ASXL2 to the dimethylated H3 lysine 4 may account for its requirement for ERα activation. On ASXL2 depletion, the proliferative potential of MCF7 cells and tumor size of xenograft mice decreased. Together with our finding on the higher ASXL2 expression in ERα-positive patients, we propose that ASXL2 could be a novel prognostic marker in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona Desmetilases/metabolismo , Humanos , Lisina/metabolismo , Células MCF-7 , Metilação , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Prognóstico , Ligação Proteica , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are caused by a high content of isoflavone, which function as partial agonists or antagonists of estrogen. To compare the estrogenic effects of SRV and soybean on the MG-63 osteoblastic cell proliferation, 70% methanol extracts of SRV or soybean were treated on MG-63 cells. Although biphasic over a concentration range of 0.001 mg/ml-0.1 mg/ml, both SRV and soybean extracts increased MG-63 cell proliferation. However SRV was more effective at increasing the cell proliferation that paralleled with the greater estrogenic effects as determined by estrogen receptor alpha (ERalpha) expression, an estrogenic response element (ERE)-luciferase activity and the selective expression of insulin-like growth factor-I (IGF-I). SRV-induced IGF-I expression resulted from increases in the mRNA levels. Despite the increased expression of ERbeta, ERE activity and IGF-I expression by soybean were lower than those by SRV. Furthermore, the comparable estrogenic effects between SRV and the combined treatment of genistein and daidzein standards at 0.5 x 10(-8) M, which is a concentration of these two isoflavones similar to that of SRV at 0.001 mg/ml, demonstrate that the greater estrogenicity of SRV for MG-63 cell proliferation is mediated by the synergism of low levels of isoflavones for the selective expression of IGF-I.
Assuntos
Estrogênios/biossíntese , Fabaceae/química , Glycine max/química , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/biossíntese , Receptor beta de Estrogênio/biossíntese , Fulvestranto , Genes Reporter/genética , Substâncias de Crescimento/biossíntese , Humanos , Fator de Crescimento Insulin-Like I/biossíntese , Luciferases/genética , Extratos Vegetais/farmacologia , Plasmídeos/genética , Receptores de Estrogênio/biossíntese , Elementos de Resposta/efeitos dos fármacos , Sementes/química , Ativação Transcricional/efeitos dos fármacosAssuntos
Antituberculosos/efeitos adversos , Eosinofilia/induzido quimicamente , Eritema/induzido quimicamente , Tuberculose Urogenital/tratamento farmacológico , Administração Tópica , Corticosteroides/administração & dosagem , Corticosteroides/uso terapêutico , Idoso , Antituberculosos/uso terapêutico , Eosinofilia/diagnóstico , Eosinofilia/imunologia , Eritema/diagnóstico , Eritema/tratamento farmacológico , Eritema/imunologia , Feminino , Antagonistas dos Receptores Histamínicos/uso terapêutico , Humanos , Testes do EmplastroRESUMO
Retinoid derivatives have been implicated for the growth regulation of ovarian cancer cells. However, the molecular mechanisms are not yet fully defined. To dissect detailed mechanisms of each derivative, four ovarian cancer cells (A2774, PA-1, OVCAR-3, SKOV-3) were treated with all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA), 13-cis RA, or 4-hydroxyphenyl retinamide (4-HPR). When treated with 1 microm, HPR inhibits most effectively the growth of all four cells. Depending on cell types treated, IC(50) values were 0.7-2.7 microm for 4-HPR, and 2.7-9.0 microm for other retinoid derivatives. DNA fragmentation assay indicated that the antiproliferative effect of HPR could be mediated by apoptosis. Transcription assays coupled with transient transfection in OVCAR-3 cells indicated that ATRA, 9-cis RA, and 13-cis RA were active for all RAR/RXR subtypes, whereas 4-HPR was only active for RARgamma. However, 4-HPR exerted the strongest suppression on AP-1 (c-Jun) activity. As expected from AP-1 data, in vitro invasion assays showed that HPR blocked effectively the migration of OVCAR-3 cells. Thus, 4-HPR showed not only more potent antiproliferative activity than any other retinoid derivatives used, but also effectively inhibited the invasion, probably through the suppression of AP-1 activity. Taken together coupled with its selective activity only for RARgamma, these results suggest that 4-HPR could be less toxic, and very effective anticancer drugs for late stage ovarian cancer.
Assuntos
Anticarcinógenos/farmacologia , Fenretinida/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Células Tumorais Cultivadas/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
The p16 gene was identified as cyclin-dependent kinase inhibitor (CDKI) and this may negatively regulate the cell cycle by acting as a tumor suppressor. Using tissue microdissection, the molecular changes at p16 and Rb genes were analysed in the spectrum of disease from dysplasia to invasive cancer of the uterine cervix. Six of 27 (22%) cases informative for D9S171 and IFNA of 9p21-22 marker (p16INK4a) showed loss of one or both alleles in at least one of these loci. LOH of pRb was detected in 29% (5/17). Gene alterations at p16 and pRb loci were only detectable in some cases of HPV-16/18 DNA positive cervical cancer. Three cases demonstrated mutational changes of p16INK4a, and the alterations were determined to be G to T shift, suggesting transitional missense mutation. In summary, the inactivation of the p16/cdk-cyclin/Rb cascade may play an additional role during the malignant progression in HPV-16/18 positive cervical cancers.
Assuntos
Carcinoma de Células Escamosas/genética , DNA de Neoplasias/química , Genes do Retinoblastoma/genética , Genes p16/genética , Perda de Heterozigosidade , Neoplasias do Colo do Útero/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Primers do DNA , DNA de Neoplasias/isolamento & purificação , DNA Viral/química , Feminino , Humanos , Invasividade Neoplásica , Papillomaviridae/genética , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
Human papillomavirus (HPV) infection is known as the major cause of the development of cervical cancer. The E6 and E7 proteins of oncogenic HPV can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, it has been determined that long-term use of oral contraceptives may be a risk factor for cervical cancer. Investigation of the estrogenic and antiestrogenic effects on the proliferation of cervical cancer cells and the gene expression of HPV would help to explain the role of estrogen in the HPV-associated pathogenesis of cervical cancer. In this study, cervical cancer cells (HeLa, CaSki, and C33A) were cultured in vitro in the presence of 17beta-estradiol or tamoxifen to observe their regulatory growth effect and HPV E6/E7 gene expression. The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient transfection assay, which was conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. The supplemental effect of estrogen receptors on URR promoter activity was also evaluated. The proliferation of HeLa and CaSki cells was stimulated by estradiol at physiologic concentration levels (=1 x 10-6 M). At a low concentration (0.1 x 10-6 M), tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, the proliferation of C33A was not influenced by exogenous estradiol or tamoxifen, indicating that HPV might play a role in the hormonal stimulation of cell growth. Interestingly, the proliferation of HeLa was markedly suppressed at high concentrations of estradiol and tamoxifen (5 and 10 x 10-6 M). The levels of HPV-18 E6 and E7 mRNA were significantly increased by estradiol at a concentration of 0.5 x 10-6 M. Transient transfection experiments using the HPV URR-CAT reporter plasmid in C33A cells indicated that the expression of HPV E6/E7 genes was increased by the treatment of estradiol and tamoxifen. Co-transfection of estrogen receptors (ER) and URR-CAT leads to a fourfold increase in CAT activity by estradiol or tamoxifen at physiologic concentrations. When estradiol or tamoxifen was administered at high concentrations (5 x 10-6 M), a DNA ladder, typically indicative of apoptosis, was observed in HeLa cells. In conclusion, estradiol stimulated the growth of HPV-positive cervical cancer cells, as did tamoxifen at low concentrations (0.1 x 10-6 M). The growth stimulation of HPV-positive cervical cancer cells by estrogen appeared to be related to the increased expression of HPV E6/E7. Growth suppression observed at high concentrations of estradiol and tamoxifen in HeLa cells might be a result of apoptosis. Taken together, these data suggested that exogenous estradiol might be a risk factor in HPV-mediated cervical carcinogenesis.
RESUMO
Human papillomavirus (HPV) DNAs are often found to be integrated into the human genome in high-grade cervical intraepithelial neoplasia (CIN) as well as in invasive cervical cancers. Investigation of the relationship between the genomic status of specific HPV genes and their antibody responses to the virus-like particles (VLPs) of HPV-16 L1/L2 proteins and the in vitro translated HPV-16 E6 and E7 proteins may help to illustrate the mechanism of HPV-related cervical carcinogenesis and host immune response. Cervical cancer tissues obtained from 39 patients were studied to evaluate the physical status of HPV genes by Southern blotting, DNA-PCR, and RT-PCR of E2. The antibody response against the HPV-16 L1/L2 VLPs of serum specimens were tested by ELISA and the antibody response against the HPV-16 E6 and E7 proteins were tested by radioimmunoprecipitation assay (RIPA), respectively. Integrated forms of HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervical cancer patients showed a significantly higher prevalence rate (39.5%; 15/38) of antibodies to HPV-16 L1/L2 VLPs than that of the control group (8.7%; 2/28) (P < 0.05). Antibodies to HPV-16 L1/L2 VLPs were more commonly detectable in cervical cancer patients having the episomal form of HPV-16 DNA (pure episomal and mixed forms) (60%; 9/15) than in those who had only the integrated forms of HPV-16 DNA (26.1%; 6/23) (P < 0.05). Antibodies to E6 and E7 proteins were positive in 36.8% (14/38) and 50% (19/38) of the patients with HPV-16 positive cervical cancer, respectively. These were significantly higher than the positive rates for the control group (8.3% and 2.8%) (P < 0.05). The differences between sero-reactivities to E6 and E7 proteins in the patients with episomal forms of HPV-16 DNA and those with integrated forms of HPV-16 DNA were not statistically significant (P > 0.05). Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer in Korea. Antibodies to HPV-16 L1/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins, appeared in a significantly larger proportion of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 L1/L2 VLPs were more often detected in cervical cancer patients having the episomal form of HPV-16 DNA than in those having only integrated forms of HPV-16 DNA. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states.
RESUMO
Although p73alpha induces many of the same cellular events as p53, it is structurally distinct from p53 in that it possesses a unique COOH-terminal domain. To dissect the function of this domain, we performed yeast two-hybrid screening of a HeLa cDNA library using residues 552-636 of p73alpha as bait. Among the clones that showed a specific interaction with p73alpha was AMP-activated protein kinase alpha (AMPKalpha). Additional yeast two-hybrid assays indicated that the betagamma-binding domain of AMPKalpha is critical for the interaction with p73alpha. The interaction was further confirmed in vitro by glutathione S-transferase pull-down, and in vivo by immunoprecipitation and immunofluorescence microscopy. Transient coexpression of AMPKalpha resulted in downregulation of the effect of p73alpha, but not of p53, on various p53-responsive promoters. Chromatin immunoprecipitation indicated p73alpha-dependent recruitment of AMPKalpha to the p21WAF1 promoter. Treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, an agonist of AMPKalpha, and expression of dominant-negative versions of AMPKalpha revealed that the repression of p73alpha was independent of AMPKalpha kinase activity. In addition, cisplatin-induced growth repression was impaired when AMPKalpha was overexpressed. Upon the knock down of AMPKalpha by siRNA, the induction of p21WAF1 by p73alpha was significantly increased. Taken together, these data indicate that AMPKalpha specifically regulates p73alpha by a direct interaction without affecting its phosphorylation status. From these data, we speculate that AMPKalpha may provide a molecular clue to understand the repressive role of the C-terminus of p73alpha in transcription and DNA damage response.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Ribonucleosídeos/farmacologia , Transfecção , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid. Herein, we investigated the antiproliferative and antiviral effects of ursolic acid and dexamethasone in human papillomavirus (HPV)-associated cervical cancer cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay to measure antiproliferative activity, and also characterized apoptosis by DNA fragmentation, 4'-6-diamidino-2-phenylindole (DAPI) staining, and flow cytometry (FACS) analysis. We investigated apoptosis-related proteins using western blots. After in vitro treatment, we used reverse transcription-polymerase chain reaction for the expression of the HPV E6/E7 gene to observe the antiviral effects. Ursolic acid suppressed the growth of HPV-positive cervical carcinoma cells (HeLa, CaSki, and SiHa) in a dose- and time-dependent manner, but not the HPV-negative cervical cancer cell line (C33A). Ursolic acid-treated HeLa cells showed typical apoptosis characteristics in DNA fragmentation, DAPI staining, and FACS analysis. The expression of Fas protein was induced, and caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP) proteins were cleaved after ursolic acid treatment. HPV-18 E6/E7 gene expression decreased after ursolic acid treatment in HeLa cells, but the levels of p53 and Rb proteins did not change. In contrast, dexamethasone, which has a similar structure, did not inhibit proliferation. Our findings may offer new insight into the mechanism of antiproliferative and antiviral effect of ursolic acid. Also, these results suggest that ursolic acid might be a useful anticancer drug in treatment of HPV-associated cervical neoplasia.
Assuntos
Antivirais/farmacologia , Dexametasona/farmacologia , Triterpenos/farmacologia , Neoplasias do Colo do Útero/patologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Antivirais/química , Antivirais/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dexametasona/química , Dexametasona/toxicidade , Feminino , Humanos , Estrutura Molecular , RNA Mensageiro/genética , Triterpenos/química , Triterpenos/toxicidade , Neoplasias do Colo do Útero/genética , Proteínas Virais/genética , Ácido UrsólicoRESUMO
Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein synthesized in a variety of normal and transformed cells, and secreted into the extracellular matrix. Based on its known effects on the tumor and endothelial cells, TSP-1 was implicated in the tumor growth and metastasis. In the present study, we have demonstrated the expression of TSP-1 in the human hepatocarcinoma cell lines. TSP-1 was detected in human hepatocarcinoma SK-HEP-1, Hep 3B and immortalized human liver Chang cells. Using two different cell lines, SK-HEP-1 and Hep 3B cells, we have studied effects of phorbol 12-myristate 13-acetate (PMA) on TSP-1 expression. TSP-1 synthesis was stimulated by PMA in both cell lines. When the cells were treated with PMA, the TSP-1 mRNA started to increase at 30 min and reached the maximal level at 6 h. TSP-1 induction by PMA was completely inhibited by the pre-treatment of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor. A TSP-1 promoter-luciferase reporter gene was transcriptionally activated by PMA, as well as by the expression of c-Jun. Among three putative AP-1 recognition sites on the TSP-1 promoter, a deletion of the 1st and 2nd sites caused loss of PMA-induced upregulation, while the 3rd site deletion showed no effect. In subsequent experiments, both the recombinant c-Jun and nuclear proteins induced by PMA have a stronger binding affinity for the 2nd AP-1 recognition site than the 1st and 3rd ones. Our study demonstrated that TSP-1 could be expressed and secreted by human hepatoma cell lines and its expression could be effectively regulated by PMA. We also suggest that AP-1 binding activity through the protein kinase C activation is a critical event for the TSP-1 gene expression and consequently affects production and processing of the protein.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Trombospondina 1/biossíntese , Trombospondina 1/genética , Fator de Transcrição AP-1/biossíntese , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Sítios de Ligação , Northern Blotting , Western Blotting , Carcinógenos , Núcleo Celular/metabolismo , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Genes Reporter , Humanos , Luciferases/metabolismo , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Acetato de Tetradecanoilforbol , Fatores de Tempo , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The bacteriophage lambda P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P x DnaB or a DnaB x DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the lambda O replication initiator enhances interaction of the P x DnaB complex with ssDNA. Partial disassembly of a ssDNA:O x P x DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.
Assuntos
Bacteriófago lambda/genética , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Reagentes de Ligações Cruzadas , DNA Helicases/metabolismo , DnaB Helicases , Modelos Genéticos , Ligação Proteica , Proteínas Virais/metabolismoRESUMO
Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution. Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors. This protein is identical to Ada3, a component of the yeast Ada coactivator complex. We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells. Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
OBJECTIVE: E6 and E7 proteins of high-risk-type human papillomavirus are major etiological agents for cervical carcinomas and are continuously expressed in those cancer cells. They inhibit cell cycle control functions by inactivating p53 and Rb proteins and also immortalize cells through the induction of telomerase activity. Expression of E6 and E7 genes in HeLa, an HPV18-positive cell line, has been shown to be inhibited by the E2 protein of bovine papillomavirus (BPV1), and this resulted in the activation of the p53-mediated growth inhibitory pathway followed by an inhibition of cell proliferation. In this study, the effect of BPV1 E2-mediated inhibition of E6 and E7 expression was examined in HPV16-positive cervical carcinoma cell lines recently established from Korean patients. METHODS: BPV1 E2 was expressed in the test cells through acute infection of an SV40-BPV1 recombinant virus. Its effect on cell proliferation was assessed through MTT and DNA synthesis assays, and the status of factors involved in cell cycle control was examined through Western blotting and reverse transcription-polymerase chain reaction. RESULTS: BPV1 E2 expression caused a significant decrease in E6/E7 transcription in all three cell lines. This was accompanied by an increase in the levels of p53 protein and activity and a decrease in the expression of Cdc25A, a Cdk2-activating phosphatase. Concomitantly, E2F1 activity and cellular DNA synthesis capacity were significantly reduced. CONCLUSIONS: These results indicate that inhibition of E6/E7 gene expression in the HPV16-positive cervical carcinoma cells induces suppression in cell proliferation by activating the growth inhibitory factors, p53 and Rb, and also by downregulating the cell cycle stimulatory factor, Cdc25A.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/fisiologia , Proteínas Oncogênicas Virais/antagonistas & inibidores , Neoplasias do Colo do Útero/virologia , Proteínas Virais/fisiologia , Animais , Papillomavirus Bovino 1/genética , Células COS , Bovinos , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Feminino , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Proteínas Oncogênicas Virais/biossíntese , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Transdução de Sinais/fisiologia , Fator de Transcrição DP1 , Fatores de Transcrição/fisiologia , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Proteínas Virais/biossíntese , Proteínas Virais/genética , Fosfatases cdc25/biossíntese , Fosfatases cdc25/genéticaRESUMO
Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.