RESUMO
AIM: To report on the multidisciplinary approach, focusing specifically on the role of the interventional radiologist (IR), used to support the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) and BATTLE-2 trials. MATERIALS AND METHODS: Patients who underwent percutaneous image-guided biopsy for the BATTLE and BATTLE-2 trials were reviewed. A radiology-based, three-point, lesion-scoring system was developed and used by two IRs. Lesions were given a score of 3 (most likely to yield sufficient material for biomarker analysis) if they met the following criteria: size >2 cm, solid mass, demonstrated imaging evidence of viability, and were technically easy to sample. Lesions not meeting all four criteria were scored 2 with the missing criteria noted as negative factors. Lesions considered to have risks that outweighed potential benefits receive a score of 1 and were not biopsied. Univariate and multivariate analyses were performed to evaluate the score's ability to predict successful yield for biomarker adequacy. RESULTS: A total of 555 biopsies were performed. The overall yield for analysis of the required biomarkers was 86.1% (478/555), and 84% (268/319) and 88.9% (210/236) for BATTLE and BATTLE-2, respectively (p=0.09). Lesions receiving a score of 3 were adequate for biomarker analysis in 89% of cases. Lesions receiving a score of 2 with more than two negative factors were adequate for molecular analysis in 69.2% (IR1, p=0.03) and 74% (IR2, p=0.04) of cases. The two IRs scored 78.4% of the lesions the same indicating moderate agreement (kappa=0.55; 95% confidence interval [CI]: 0.48, 0.61). CONCLUSIONS: IRs add value to clinical trial teams by optimising lesions selected for biopsy and biomarker analysis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Radiologia Intervencionista/métodos , Idoso , Biópsia por Agulha Fina , Ensaios Clínicos como Assunto , Feminino , Humanos , Biópsia Guiada por Imagem , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Equipe de Assistência ao PacienteRESUMO
We evaluated whether active osteoporosis care in patients experiencing their first distal radius fracture (DRF) reduces subsequent hip or spine fractures by comparing two cohorts. The incidence of subsequent fractures was significantly lower in the active care cohort than the other cohort in 4-year follow-up. PURPOSE: Studies show that osteoporosis care in patients with osteoporotic fracture reduces subsequent fractures, but the impact of such active care in patients with distal radius fracture (DRF) has not been well studied. We evaluated how much osteoporosis care in patients experiencing their first DRF can reduce subsequent hip or spine fractures at 4-year follow-up. METHODS: Active osteoporosis care by orthopedic surgeons for patients with DRF started from September 2009 at our institution, thus we had a unique opportunity to compare the two cohorts: pre-involvement (PreI) group (DRF before September 2009) and post-involvement (PostI) group (DRF from September 2009). We compared the two cohorts for subsequent hip or spine fracture incidence in the 4 years following DRF. RESULTS: Overall, 1057 patients with a DRF (85% women; mean age, 70 years) were studied, of whom 205 patients were in PreI group and 852 in PostI group. Subsequent fractures occurred in 27 patients (2.6%), with a mean interval of 29 months after DRF. The incidence was significantly lower in the PostI group than in the PreI group (1.9% vs. 5.4%, p = 0.004), especially in hip fractures (0.4% vs. 2.9%, p = 0.002). The relative risk reduction was 65% for all subsequent fractures and 86% for hip fractures. CONCLUSION: This study demonstrates that active osteoporosis care in patients with DRF significantly reduces subsequent fracture incidence even for the 4-year follow-up period. These findings add an evidence for the current proactive osteoporosis care programs such as fracture liaison services. LEVEL OF EVIDENCE: Therapeutic level III.
Assuntos
Fraturas do Quadril , Osteoporose , Fraturas por Osteoporose , Fraturas do Rádio , Fraturas da Coluna Vertebral , Idoso , Feminino , Fraturas do Quadril/epidemiologia , Fraturas do Quadril/etiologia , Fraturas do Quadril/prevenção & controle , Humanos , Estudos Longitudinais , Masculino , Osteoporose/complicações , Osteoporose/epidemiologia , Osteoporose/terapia , Fraturas por Osteoporose/epidemiologia , Fraturas por Osteoporose/etiologia , Fraturas por Osteoporose/prevenção & controle , Fraturas do Rádio/complicações , Fatores de Risco , Fraturas da Coluna Vertebral/epidemiologia , Fraturas da Coluna Vertebral/etiologia , Fraturas da Coluna Vertebral/prevenção & controleRESUMO
BACKGROUND: The aim of this study is to investigate the prognostic role of phosphorylated AMP-activated protein kinase (pAMPK) in surgically resected non-small-cell lung cancer (NSCLC). METHODS: Immunohistochemical staining of pAMPK was carried out on tissue microarrays containing 463 samples obtained from patients with NSCLC and correlated with clinicopathological characteristics and survival. RESULTS: pAMPK expression levels were significantly higher in never smokers versus former smokers versus current smokers (P=0.045). A positive pAMPK expression was associated with increased overall survival (OS) and recurrence-free survival (RFS) (P=0.0009 and P=0.0007, respectively). OS and RFS were statistically superior in pAMPK-positive than in pAMPK-negative patients with adenocarcinoma (ADC; median OS: 5.6 and 4.2 years, respectively, P=0.0001; median RFS: 5.0 and 2.4 years, respectively, P=0.001), whereas they were similar in those patients with squamous cell carcinoma. Multivariate analysis confirmed that pAMPK positivity was associated with OS [hazard ratio (HR)=0.574, 95% confidence interval (CI) 0.418-0.789, P=0.0006) and RFS (HR=0.608, 95% CI 0.459-0.807, and P=0.0006), independent of clinical covariates. CONCLUSIONS: High pAMPK expression levels are associated with increased survival in patients with NSCLC, especially those with ADC. Our results support further evaluation of AMP-activated protein kinase as a potential prognostic and therapeutic target for lung cancer.
Assuntos
Proteínas Quinases Ativadas por AMP/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Modelos de Riscos Proporcionais , Análise Serial de TecidosAssuntos
Eritema/etiologia , Dermatoses Faciais/etiologia , Dor Facial/etiologia , Herpes Zoster Oftálmico/complicações , Hemorragias Intracranianas/etiologia , Aciclovir/administração & dosagem , Antivirais/administração & dosagem , Eritema/tratamento farmacológico , Dermatoses Faciais/tratamento farmacológico , Dor Facial/tratamento farmacológico , Herpes Zoster Oftálmico/líquido cefalorraquidiano , Herpes Zoster Oftálmico/tratamento farmacológico , Humanos , Imunocompetência , Hemorragias Intracranianas/líquido cefalorraquidiano , Hemorragias Intracranianas/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Radiografia , Resultado do TratamentoAssuntos
Lipoma/diagnóstico , Neoplasias Musculares/diagnóstico , Músculos do Pescoço/patologia , Diagnóstico Diferencial , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Lipoma/patologia , Masculino , Processo Mastoide , Pessoa de Meia-Idade , Neoplasias Musculares/patologia , EsternoRESUMO
AIMS: To evaluate a traditional yoghurt used as folk medicine for its ability to kill Helicobacter pylori in vitro. METHODS AND RESULTS: Micro-organisms from the yoghurt were identified and tested in different food substrates for their effects on H. pylori in a co-culture well system. Two yeasts and several strains of lactobacilli were isolated from the yoghurt, and both the yeast and the lactobacilli independently showed cidal activity against H. pylori. The microbes from the original yoghurt also retained their cidal effect when grown in corn meal and soy milk. CONCLUSIONS: The yeast and lactobacilli found in this yoghurt form a hardy symbiotic culture. The organisms secrete soluble factors capable of killing H. pylori, and these factors may include some organic by-products of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: These yoghurt-derived food preparations could become simple and inexpensive therapies to suppress H. pylori infections in endemic countries.
Assuntos
Helicobacter pylori , Medicina Tradicional , Probióticos , Iogurte/microbiologia , Técnicas Bacteriológicas , Técnicas de Cocultura , Infecções por Helicobacter/terapia , Humanos , Lactobacillus , Glycine max , Simbiose , Leveduras , Zea maysRESUMO
A prospective randomized study was conducted to determine whether amifostine (Ethyol) reduces the rate of severe esophagitis and hematologic and pulmonary toxicity associated with chemoradiation or improves control of non-small cell lung cancer (NSCLC). Sixty patients with inoperable stage II or III NSCLC were treated with concurrent chemoradiotherapy. Both groups received thoracic radiation therapy (TRT) with 1.2 Gy/fraction, 2 fraction per day, 5 days per week for a total dose 69.6 Gy. All patients received oral etoposide (VP-16), 50 mg Bid, 30 minutes before TRT beginning day 1 for 10 days, repeated on day 29, and cisplatin 50 mg/m(2) intravenously on days 1, 8, 29, and 36. Patients in the study group received amifostine, 500 mg intravenously, twice weekly before chemoradiation (arm 1); patients in the control group received chemoradiation without amifostine (arm 2). Patient and tumor characteristics were distributed equally in both groups. Of the 60 patients enrolled, 53 were evaluable (27 in arm 1, 26 in arm 2) with a median follow-up of 6 months. Median survival times were 26 months for arm 1 and 15 months for arm 2, not statistically significantly different. Morphine intake to reduce severe esophagitis was significantly lower in arm 1 (2 of 27, 7.4%) than arm 2 (8 of 26, 31%; P =.03). Acute pneumonitis was significantly lower in arm 1 (1 of 27, 3.7%) than in arm 2 (6 of 26, 23%; P =.037). Hypotension (20 mm Hg decrease from baseline blood pressure) was significantly more frequent in arm 1 (19 of 27, 70%) than arm 2 (1 of 26, 3.8%; P =.0001). Only 1 patient discontinued treatment because of hypotension. These preliminary results showed that amifostine significantly reduced acute severe esophagitis and pneumonitis. Further observation is required to assess long-term efficacy.
Assuntos
Amifostina/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Etoposídeo/uso terapêutico , Neoplasias Pulmonares/radioterapia , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Terapia Combinada , Esofagite/etiologia , Esofagite/prevenção & controle , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Pneumonite por Radiação/prevenção & controleRESUMO
BACKGROUND: Abnormalities of PTEN, a candidate tumor suppressor gene located at 10q23.3, play an important role in the tumorigenesis of multiple tumor types. OBJECTIVES: To investigate the expression of PTEN and its clinical implication in squamous cell carcinoma of the tongue. DESIGN: Retrospective analysis of PTEN protein expression in archived primary oral tongue tumor samples. SETTING: Academic center. PATIENTS AND METHODS: PTEN expression was determined by immunohistochemical analysis in tissue samples from 41 patients with stage II, III, and IV squamous cell carcinoma of the tongue. All the patients underwent curative surgical treatment with a median follow-up of 81 months. The Kaplan-Meier method was used for survival analysis. Multivariate analysis was performed according to the Cox proportional hazards model. RESULTS: Lack of staining for PTEN was demonstrated in 12 (29%) of the 41 tumors. Patients whose tumors lacked PTEN expression had a significantly shorter overall survival time (P = .03) and event-free survival time (P = .01) than those patients with positive PTEN expression. Multivariate regression analysis demonstrated that PTEN expression is an independent predictor of poor outcome when compared with tumor stage and nodal status. CONCLUSIONS: Although genetic alterations of the PTEN gene are rare in head and neck squamous cell carcinoma, loss of PTEN is not an uncommon event in squamous cell carcinoma of the tongue. Lack of PTEN expression may be an independent prognostic indicator for clinical outcome in patients with this tumor type.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Genes Supressores de Tumor , Monoéster Fosfórico Hidrolases/genética , Neoplasias da Língua/diagnóstico , Proteínas Supressoras de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Feminino , Marcadores Genéticos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias da Língua/genética , Neoplasias da Língua/mortalidadeRESUMO
BACKGROUND: Thalidomide has been shown to have antiangiogenic effects in preclinical models as well as a significant antitumor effect in hematologic tumors such as multiple myeloma. The authors performed this Phase II study to determine the activity, toxicity profile, and antiangiogenic effect of thalidomide in patients with locoregionally recurrent or metastatic squamous cell carcinoma of the head and neck. METHODS: Twenty-one patients with recurrent or metastatic squamous cell carcinoma of the head and neck were treated with single-agent thalidomide. All patients had received radiation therapy, and most had undergone surgery (95%) and/or chemotherapy (90%). Thalidomide was initiated at 200 mg;3>daily and increased to a target dose of 1000 mg daily. Patients continued treatment until disease progression, unacceptable toxicity, or death occurred. RESULTS: All 21 patients eventually developed progressive disease. Median time to progression was 50 days (95% confidence interval, 28-70), with median overall survival time of 194 days (95% lower confidence boundary, 151), similar to the progression and survival times reported for this patient group with other agents. Thalidomide was generally well tolerated, with few patients experiencing Grades 3 to 4 toxicities. Serum vascular endothelial growth factor and basic fibroblast growth factor levels increased in six of seven patients, for whom paired serum samples were available and all of whom had progressive disease. CONCLUSIONS: In this heavily pretreated population of patients with advanced squamous cell carcinoma of the head and neck, thalidomide does not appear to have single-agent antitumor activity. Further evaluation of the mechanism of action of thalidomide is indicated. Potentially, future evaluations of thalidomide may be performed in combination with other antiangiogenic or cytotoxic agents in patients with earlier stage disease or in patients with minimal residual disease.
Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Talidomida/farmacologia , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/efeitos adversos , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Talidomida/efeitos adversos , Resultado do TratamentoRESUMO
The melanoma antigen (MAGE)-encoding genes are expressed in various tumor types, including lung, and are thought to be silent in all normal tissues except testis. In search of biomarkers for early lung cancer detection and cancer risk assessment, we investigated frequencies of expressional activation of MAGE-A1, -A3, and -B2 genes in non-small cell lung cancers (NSCLCs). Expression of these genes was evaluated by reverse transcription-PCR (RT-PCR) in 20 primary NSCLC samples and corresponding normal lung tissues as well as in 20 bronchial brush specimens from former smokers without lung cancer. mRNA in situ hybridization was done to confirm the gene expression pattern at the cellular level. Methylation-specific PCR was performed to evaluate the hypomethylation status of CpG sites in the promoter regions of these genes. Among the 20 primary NSCLC samples analyzed, 14 (70%) expressed MAGE-A1 and 17 (85%) each expressed MAGE-A3 and MAGE-B2. A substantial number of normal lung tissues adjacent to NSCLC also had a detectable level of MAGE expression (65, 75, and 80% for MAGE-A1, -A3, and -B2, respectively). We found that 7 (35%), 10 (50%), and 11 (55%) of the adjacent normal lung tissue samples exhibited promoter hypomethylation at MAGE-A1, -A3, and -B2, respectively, compared with 15 (75%), 16 (80%), and 16 (80%) of the NSCLC samples. Among the 20 bronchial epithelium samples from former smokers, 7 (35%), 10 (50%), and 12 (60%) had also detectable -A1, -A3, and -B2 expression, respectively. Activation of MAGE-A1, -A3, and -B2 genes is common not only in NSCLC but also in bronchial epithelium with severe carcinogen insult. These results suggest that MAGE genes may be activated very early in lung carcinogenesis and may be considered as targets for lung cancer prevention.
Assuntos
Antígenos de Neoplasias/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Idoso , Antígenos de Neoplasias/biossíntese , Ilhas de CpG , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Antígenos Específicos de Melanoma , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Previous trials of topical trans-retinoic acid treatment of cervical intraepithelial neoplasia (CIN) grades 2 and 3 led to a statistically significant regression of CIN 2, but not CIN 3. We tested N-(4-hydroxyphenyl)retinamide (4-HPR), a promising oral retinoid that has been shown to induce apoptosis through nonretinoic receptor acid-mediated pathways, for its toxicity and efficacy against CIN 2/3. EXPERIMENTAL DESIGN: In a blinded randomized trial, 4-HPR at 200 mg/day for 6 months (with a 3-day/month drug holiday) was compared with placebo in patients with biopsy-proven CIN-2/3 [high-grade squamous intraepithelial lesions (HGSILs)]. Patients were treated with placebo or 4-HPR for 6 months, biopsied, and then followed for an additional 6 months. At the 12-month end point, they underwent either loop excision if a histological lesion was present or a biopsy from the original area of the lesion if no lesion was present. RESULTS: An interim analysis of blinded data showed a significantly worse prognosis at 12 months for one group. When the code was broken because of the poorer outcomes, we discovered that the 4-HPR treatment arm was performing more poorly than was the placebo at 6 and 12 months (25 versus 44% response rates at 6 months; 14 versus 50% at 12 months). Toxicity was not significant in either arm. CONCLUSIONS: 4-HPR at 200 mg/day with a 3-day/month drug holiday is not active compared with placebo in the treatment of HGSIL. Because 4-HPR is active in the laboratory, the lack of effect in our trial may indicate that higher doses are needed in patients to achieve comparable results.
Assuntos
Antineoplásicos/uso terapêutico , Fenretinida/uso terapêutico , Displasia do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Adulto , Antineoplásicos/efeitos adversos , Queilite/induzido quimicamente , Estudos Cross-Over , Exantema/induzido quimicamente , Feminino , Fenretinida/efeitos adversos , Fenretinida/sangue , Humanos , Futilidade Médica , Cooperação do Paciente , Transtornos de Fotossensibilidade/induzido quimicamente , Fatores de Tempo , Resultado do Tratamento , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/patologiaRESUMO
Cisplatin-based chemotherapy improves survival in appropriately selected patients with stage IV non-small cell lung cancer (NSCLC). However, cisplatin-based regimens have well-known dose-related toxicities, particularly renal insufficiency and neurotoxicity. On the basis of prior preclinical and phase I studies, we initiated a phase II study of SPI-77 (STEALTH) Liposomal Cisplatin) in patients with stage IIIB and IV NSCLC who failed previous treatment with platinum. Disease in all subjects had progressed during therapy, failed to respond, or progressed within 3 months after discontinuing the platinum-based chemotherapy. Between January and June 1999, 13 patients were enrolled at our institution. Patient characteristics included: seven women, six men; median age, 61 years; median Karnofsky performance status, 80%; median number of prior chemotherapy regimens, two (range, 1-3). All patients had adequate hepatic and renal function. SPI-77 was administered at a dose of 260 mg/m(2) IV every 3 weeks. A median of two cycles (range 1-6) were given; the total number of cycles was 35. Among the 12 patients evaluable for response, two had (17%) stable disease and ten (83%) had progressive disease. The median survival was 24.3 weeks, and the median follow-up was 43.9 weeks. Toxicity could be evaluated in all subjects. Moderate anemia (46% of cycles,
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Progressão da Doença , Feminino , Humanos , Lipossomos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida , Resultado do TratamentoRESUMO
PURPOSE: To better understand the role of G(1)-S transition regulator abnormalities in the pathogenesis of advanced premalignant lesions of the upper aerodigestive tract and the biological effects of chemoprevention, we studied biopsies obtained sequentially from participants in a prospective trial using 13-cis retinoic acid, IFN-alpha, and alpha-tocopherol for 12 months. EXPERIMENTAL DESIGN: Cyclin D1 and p16 expression were analyzed by immunohistochemistry, loss of heterozygosity by polymerase chain reacting amplification, and then electrophoretic separation of the products, methylation of the p16 promoter by methylation-specific polymerase chain reacting, and cyclin D1 gene amplification by fluorescence in situ hybridization. RESULTS: Baseline dysregulation of cyclin D1 expression was found in 50% (14 of 28) and was reversed in 6 of 14 cases, whereas p16 expression was lost in 46% (13 of 28) and regained in 2 of 13 cases. Loss of heterozygosity at 9p21 occurred in 68% and p16(INK4a) promoter methylation occurred in 75% of cases, with increasing frequency from mild to severe dysplasia. Cyclin D1 gene amplification was identified in two cases. Cyclin D1 protein dysregulation at last follow-up alone and in combination with p16 loss was associated with histological progression and cancer development (P < 0.01). CONCLUSIONS: Additional study of these alterations in a larger sample and exploration of the upstream signaling partners of these cell cycle regulators in vivo is warranted to identify cancer risk profiles that would be meaningful targets for chemopreventive intervention.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Ciclinas/genética , Neoplasias de Cabeça e Pescoço/patologia , Lesões Pré-Cancerosas/patologia , Adulto , Idoso , Proteínas de Ciclo Celular/fisiologia , Cromossomos Humanos Par 9/genética , Ciclina D , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , DNA/genética , DNA/metabolismo , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/prevenção & controle , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismoRESUMO
The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.
Assuntos
Actinas/genética , Proteínas Fúngicas/genética , Pichia/genética , Regiões Promotoras Genéticas , Seleção Genética , Actinas/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Proteínas Fúngicas/química , Genes Fúngicos , Higromicina B/farmacologia , Íntrons , Dados de Sequência Molecular , Compostos Organoplatínicos , Oxaliplatina , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Pichia/efeitos dos fármacos , Pichia/metabolismo , Splicing de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Transformação GenéticaRESUMO
BACKGROUND: Telomerase activation plays a critical role in tumorigenesis. To determine the role of telomerase in early lung carcinogenesis and as a potential biomarker in chemoprevention trials, we analyzed the expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens from cigarette smokers who were enrolled in a randomized, double-blinded, placebo-controlled chemoprevention trial of N-(4-hydroxyphenyl)retinamide (4-HPR). METHODS: We obtained biopsy specimens from six predetermined sites in the bronchial tree from the 57 participants, before treatment and 6 months after treatment with 4-HPR or placebo. We used in situ hybridization to examine hTERT messenger RNA (mRNA) expression in 266 pretreatment (baseline) and post-treatment site-paired biopsy specimens from 27 patients in the 4-HPR-treated group and from 30 patients in the placebo-treated group. All statistical tests were two-sided. RESULTS: At baseline, 62.4% (95% confidence interval [CI] = 53.9% to 71%) of the biopsy specimens obtained from the group treated with 4-HPR and 65.2% (95% CI = 57.4% to 73.1%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. After 6 months, 45.6% (95% CI = 36.9% to 54.3%) of the biopsy specimens obtained from the 4-HPR-treated group and 68.1% (95% CI = 60.4% to 75.8%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. The reduction in hTERT expression observed between the two treatment groups over time was statistically significant (P =.01) when we used the biopsy site as the unit of analysis, but not when we used the individual as the unit of analysis (P =.37). CONCLUSIONS: Telomerase is frequently reactivated in the lungs of cigarette smokers. The modulation of hTERT expression in 4-HPR-treated smokers suggests that a novel molecular mechanism underlies the potential chemopreventive properties of 4-HPR. hTERT expression is a promising potential biomarker for risk assessment and for the evaluation of the efficacy of chemopreventive agents in lung carcinogenesis.
Assuntos
Anticarcinógenos/farmacologia , Biomarcadores Tumorais/metabolismo , Brônquios/enzimologia , Fenretinida/farmacologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/prevenção & controle , Fumar/metabolismo , Telomerase/efeitos dos fármacos , Telomerase/metabolismo , Adulto , Idoso , Brônquios/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Proteínas de Ligação a DNA , Método Duplo-Cego , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Pulmonares/etiologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Neoplásico/análise , Medição de Risco , Fumar/efeitos adversos , Telomerase/genética , Resultado do TratamentoRESUMO
To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.