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1.
Ann R Coll Surg Engl ; 98(3): 192-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26876538

RESUMO

INTRODUCTION: Retroperitoneal sarcoma is a surgically managed condition that can recur locally following macroscopically complete resection. Owing to the low incidence of the condition, advances in treatment are reported infrequently but complete compartmental resection and adjuvant or neoadjuvant radiotherapy are areas under investigation. Given the practical difficulty of randomised trials, observational data can highlight advantages from progressive treatment approaches. METHODS: A retrospective database of consecutive retroperitoneal sarcoma resections performed at a single referral centre between March 1997 and March 2013 was interrogated. Histological, radiological and clinical data were collected. Univariate and multivariate analyses for disease free and overall survival were performed to establish independent predictors of disease recurrence and patient survival. RESULTS: A total of 79 patients underwent 90 resections (63 primary). The mean five-year overall and disease free survival rates were 55.3% and 24.8% respectively. Higher patient age, high tumour grade, presence of extraretroperitoneal disease and invasive tumour phenotype were found to significantly predict survival following multivariate analysis. Half (50%) of the tumours displayed invasive behaviour on histopathology and 42% of locoregional recurrence was intraperitoneal. CONCLUSIONS: Retroperitoneal sarcoma is commonly an infiltrative tumour and often recurs outside of the retroperitoneum. These features limit the therapeutic impact of interventions that focus on gaining local control such as complete compartmental resection and radiotherapy. It seems likely that future advances in the management of this cancer will involve new systemic agents to treat this frequently systemic disease.


Assuntos
Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/cirurgia , Neoplasias Retroperitoneais/epidemiologia , Neoplasias Retroperitoneais/cirurgia , Sarcoma/epidemiologia , Sarcoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Neoplasias Retroperitoneais/patologia , Estudos Retrospectivos , Sarcoma/patologia , Resultado do Tratamento , Adulto Jovem
2.
Br J Cancer ; 112(2): 290-5, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25356804

RESUMO

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.


Assuntos
Neuroblastoma/genética , Neoplasias do Sistema Nervoso Periférico/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Intervalo Livre de Doença , Amplificação de Genes , Humanos , Lactente , Estimativa de Kaplan-Meier , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/diagnóstico , Neuroblastoma/mortalidade , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Neoplasias do Sistema Nervoso Periférico/diagnóstico , Neoplasias do Sistema Nervoso Periférico/mortalidade , Prognóstico
3.
Br J Cancer ; 111(4): 716-25, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-24921920

RESUMO

BACKGROUND: A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor. METHODS: This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis. RESULTS: Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines. CONCLUSIONS: These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Concentração Inibidora 50 , Neuroblastoma/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirrolidinas/farmacologia , Compostos de Espiro/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Verapamil/farmacologia , Vincristina/metabolismo , Vincristina/farmacologia , para-Aminobenzoatos/farmacologia
4.
Oncogene ; 31(6): 752-63, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21725357

RESUMO

MYCN amplification is a major biomarker of poor prognosis, occurring in 25-30% of neuroblastomas. MYCN has contradictory roles in promoting cell growth and sensitizing cells to apoptosis. We have recently shown that p53 is a direct transcriptional target of MYCN in neuroblastoma and that p53-mediated apoptosis may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14(ARF) pathway is often inactivated through MDM2 amplification or p14(ARF) inactivation. We hypothesized that reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis especially in MYCN-amplified cells. Using the SHEP Tet21N MYCN-regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared with MYCN(+) cells and siRNA-mediated knockdown of MYCN in four MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of 18 neuroblastoma cell lines treated with Nutlin-3 and MI-63, the subset amplified for MYCN had a significantly lower mean GI(50) value (50% growth inhibition) and increased caspase 3/7 activity compared with the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. We conclude that amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wild-type p53 to MDM2-p53 antagonists and that these compounds may therefore be particularly effective in treating high-risk MYCN-amplified disease.


Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Piperazinas/farmacologia , Compostos de Espiro/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Modelos Biológicos , Mutação , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Tetraciclina/farmacologia , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Br J Cancer ; 102(3): 553-60, 2010 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-20051956

RESUMO

BACKGROUND: The standard treatment of choice for malignant pleural mesothelioma is chemotherapy with pemetrexed and platinum, but the clinical outcome is poor. This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FRalpha). METHODS: Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50% (GI(50)). FRalpha expression was determined by western blotting and that of FRalpha, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT-PCR. Immunohistochemistry for FRalpha was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed. RESULTS: A wide range of GI(50) values was obtained for the cell lines, H2452 cells being the most sensitive (GI(50) 22 nM) and RS5 cells having a GI(50) value greater than 10 microM. No FRalpha protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRalpha was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRalpha and the outcome of pemetrexed treatment, and no significant difference between histological subtypes. CONCLUSION: Response to treatment with pemetrexed does not depend on the presence of FRalpha.


Assuntos
Proteínas de Transporte/fisiologia , Antagonistas do Ácido Fólico/uso terapêutico , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Receptores de Superfície Celular/fisiologia , Western Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Receptores de Folato com Âncoras de GPI , Guanina/uso terapêutico , Humanos , Imuno-Histoquímica , Pemetrexede , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Regul Toxicol Pharmacol ; 53(2): 107-20, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19027814

RESUMO

In response to a Hazard Notice by the Medical Devices Agency of the UK in 2000 regarding the Trilucent breast implant (TBI), an expert panel was convened to implement a research program to determine whether genotoxic compounds were formed in the soybean oil filler (SOF) of TBIs and whether these could be released to produce local or systemic genotoxicity. The panel established a research program involving six laboratories. The program recruited 47 patients who had received TBIs (9 patients had received silicone implants previously). A reference group (REBI) of 34 patients who had exchanged either silicone (17 patients) implants (REBI-E) or patients (17) who were to receive primary implantation augmentation with silicone (REBI-PIA), and who were included as needed to increase either the pre- or post-explantation sample number. Of the 17 REBI-E patients, 5 had silicone implants and 12 had saline implants previously (prior to the last exchange). Investigation was undertaken before and after replacement surgery in the TBI patients and before and after replacement or augmentation surgery in the REBI patients. The pre- to post-operative sample interval was 8-12 weeks. Pre-operative samples were collected within 7 days prior to the operation. Information on a variety of demographic and behavioral features was collected. Biochemical and biological endpoints relating to genotoxic lipid peroxidation (LPO) products potentially formed in the SOF, and released locally or distributed systemically, were measured. The SOF of explanted TBIs was found to have substantial levels of LPO products, particularly malondialdehyde (MDA), and low levels of trans-4-hydroxy-2-nonenal (HNE) not found in unused implants. Mutagenicity of the SOF was related to the levels of MDA. Capsules that formed around TBIs were microscopically similar to those of reference implants, but MDA-DNA adducts were observed in capsular macrophages and fibroblasts of only TBI capsules. These cell types are not progenitors of breast carcinoma (BCa) and the location of the implants precludes LPO products reaching the mammary epithelial cells which are progenitors of BCa. Blood levels of LPO products were not increased in TBI patients compared to REBI patients and did not change with explantation. In TBI patients, white blood cells did not show evidence of increased levels of LPO-related aldehyde DNA adducts. In conclusion, based on a number of measured parameters, there was no evident effect that would contribute to breast or systemic cancer risk in the TBI patients, and the recommended treatment of TBI patients involving explantation was judged appropriate.


Assuntos
Implantes de Mama/efeitos adversos , Peroxidação de Lipídeos , Testes de Mutagenicidade , Óleo de Soja/efeitos adversos , Adulto , Aldeídos/metabolismo , Remoção de Dispositivo , Feminino , Fibroblastos/metabolismo , Humanos , Macrófagos/metabolismo , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Falha de Prótese , Géis de Silicone , Cloreto de Sódio/química
7.
J Clin Pathol ; 61(10): 1098-103, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18682419

RESUMO

BACKGROUND: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. AIMS: To report the histopathological features of neuroblastomas from MYCN transgenic mice. METHODS: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. RESULTS: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. CONCLUSIONS: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.


Assuntos
Neoplasias Abdominais/patologia , Neuroblastoma/patologia , Proteínas Nucleares , Proteínas Oncogênicas , Neoplasias Abdominais/genética , Animais , Biomarcadores/análise , Western Blotting , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Masculino , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Oncogênicas/análise , Proteínas Oncogênicas/genética , Proto-Oncogene Mas , Ubiquitina Tiolesterase/análise
8.
Colorectal Dis ; 10(4): 344-51, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17949449

RESUMO

OBJECTIVE: Identification of biological markers that may predict response to chemotherapy would allow the individualization of treatment by enabling selection of patients most likely to benefit from chemotherapy. The aims of this study were to determine whether p53 mutation status and p53 and p33(ING1b) protein expression can predict which patients with Dukes' C colorectal cancer following curative surgical resection respond to adjuvant chemotherapy with 5-fluorouracil (5-FU). METHOD: Patients with Dukes'C colorectal cancer (n = 41) were studied. DNA was extracted and analysed for p53 mutation using PCR-based direct DNA sequencing. Tumours were analysed for p53 protein expression by immunohistochemistry using DO-7 monoclonal antibody and for p33(ING1b) protein expression using GN1 monoclonal antibody. RESULTS: There was a significant association between p53 mutation status analysed by gene sequencing and overall and metastasis-free survival (P = 0.03 and 0.004, respectively, log-rank test). By contrast, no significant correlation was found between p53 and p33(ING1b) protein expression and overall or metastasis-free survival. CONCLUSION: In patients with Dukes'C colorectal cancer who underwent curative surgical resection of the primary tumour, followed by 5-FU-based adjuvant chemotherapy, p53 mutation status as assessed by gene sequencing is a significant predictor of overall and metastasis-free survival.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Quimioterapia Adjuvante/métodos , Estudos de Coortes , Neoplasias Colorretais/classificação , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Fluoruracila/uso terapêutico , Humanos , Proteína 1 Inibidora do Crescimento , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação/genética , Valor Preditivo dos Testes
9.
Br J Cancer ; 96(5): 762-8, 2007 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-17311025

RESUMO

The effect of EGF and gefitinib on two EGFR-positive human bladder cancer cell lines has been investigated using array-based gene expression profiling. The most prominent transcript, increased up to 6.7-fold by EGF compared with controls in RT112 cells, was human early growth response protein 1 (hEGR1). This induction was prevented by gefitinib. The hEGR1 mRNA in EGF-treated samples was reduced in the presence of gefitinib, as was hEGR1 protein in cell lysates. In the RT4 cells, hEGR1 expression was halved in the presence of EGF and gefitinib in combination. In bladder tumour samples, there was a significant correlation between hEGR1 mRNA detected by RT-PCR and EGFR detected by ligand binding, (P=0.042). The induction by EGF of the hEGR1 gene, mRNA and protein in RT112 cells, and its inhibition by gefitinib, together with the detection of hEGR1 mRNA in bladder tumours, suggests that hEGR1 may be important in the EGFR growth-signalling pathway in bladder cancer and should be further investigated for its prognostic significance and as a potential therapeutic target.


Assuntos
Antineoplásicos/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Quinazolinas/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Gefitinibe , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Urology ; 66(1): 196-200, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15992892

RESUMO

OBJECTIVES: To investigate expression patterns of erbB receptors in a panel of 58 human bladder tumors. Aberrant functional and structural interactions of erbB type I receptor tyrosine kinase cell surface receptors are important in the development and maintenance of the malignant phenotype. Few studies have focused on the remaining family members or patterns of receptor coexpression in urothelial cancer. METHODS: Frozen tumor samples from 58 patients with newly diagnosed bladder cancer were collected; 18 had Stage Ta, 20 Stage T1, and 20 had Stage T2 or worse. The grade was G1 in 5, G2 in 24, and G3 in 29 patients. Seven normal urothelial samples were obtained from patients with benign urologic conditions. The tumor material was probed using conventional immunoblotting and enhanced chemiluminescence. The blots were captured with digital imaging, and protein expression was quantified with gel analysis software. RESULTS: Most tumors exhibited detectable expression of at least one erbB receptor. Examples of coexpression of epidermal growth factor receptor (EGFr) and erbB-2 were also found. Detectable erbB-3 or erbB-4 protein expression was lacking in this series. Compared with other tumors, the T1 samples exhibited the greatest mean levels of erbB-2 protein expression (P = 0.0028). Of the 58 tumors, 10 (17.2%) coexpressed EGFr and erbB-2; this was associated with T1 disease (P = 0.03). CONCLUSIONS: Varied levels of expression of both EGFr and erbB-2 appear to exist in human bladder cancer. These preliminary data do not support erbB-3/4 as major protagonists in this tumor system. The observations presented suggest a role for EGFr and erbB-2 in the development and progression of bladder cancer that should be explored further.


Assuntos
Receptor ErbB-2/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Receptores ErbB/biossíntese , Feminino , Humanos , Masculino , Neoplasias da Bexiga Urinária/patologia
11.
Br J Cancer ; 90(8): 1679-85, 2004 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-15083203

RESUMO

The effect of EGF stimulation and its inhibition with gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been investigated in two EGFR-positive human bladder tumour cell lines, RT112 and RT4. The growth of RT112 cells in a medium containing 10% foetal bovine serum was inhibited by 50% with 10 microM gefitinib, whereas this dose completely inhibited RT4 cell growth. Cells were more sensitive to growth inhibition in the serum-free medium. Increased growth of cells in the serum-free medium was observed with 10 or 50 ng x ml(-1) EGF and the proliferative effect of EGF stimulation in both cell lines was inhibited in the presence of 1 microM, but not 0.1 microM gefitinib. Zymography of the conditioned medium from RT112 cells treated with EGF and gefitinib showed a decrease in matrix metalloproteinase 2 (MMP2) concentrations. Western blot analysis showed that tissue inhibitor of metalloproteinase 1(TIMP1) increased in the conditioned medium from RT112 cells treated with EGF, and this was partially inhibited with both 1 and 5 microM gefitinib. Conversely, TIMP2 decreased with EGF stimulation and this was reversed with gefitinib. Tissue inhibitor of metalloproteinase 1 had no effect on the growth of either cell line. These studies show alterations in the balance of MMPs and their inhibitors in EGF-stimulated bladder tumour cells, which are reversed by gefitinib, suggesting gefitinib should be investigated for its effect on human bladder tumours.


Assuntos
Antineoplásicos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/fisiologia , Inibidores de Proteases/análise , Quinazolinas/farmacologia , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Neoplasias da Bexiga Urinária/patologia , Gefitinibe , Humanos , Células Tumorais Cultivadas
12.
J Clin Pathol ; 56(7): 491-6, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12835293

RESUMO

The inhibitor of growth (ING) genes (ING1-4) probably descend from tumour suppressor genes. ING1 was the first to be identified and later isolated using an approach to detect genes whose expression is suppressed in cancer. The others were isolated through homology and similarity searches in human and mouse databases. All members contain a plant homeodomain involved in macromolecule recognition. Apart from the extensively studied ING1, little is known about the number of transcripts encoded by the other members or their gene structure. ING1 encodes several differentially spliced mRNAs, which may produce a family of proteins. The most widely expressed protein isoform is p33(INGb1), which is involved in restriction of cell growth and proliferation, apoptosis, tumour anchorage independent growth, cellular senescence, maintenance of genomic stability, and modulation of cell cycle checkpoints. ING1 gene mutation is uncommon in cancer, although the subcellular localisation of p33(INGb1) may have an effect on its function. The p33(INGb1) cellular compartmental shift from the nucleus to the cytoplasm may cause loss of normal cellular function, and may play a central role in the pathogenesis of several cancers.


Assuntos
Genes Supressores de Tumor , Neoplasias/genética , Proteínas/genética , Apoptose/genética , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Cromatina , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Genes p53 , Predisposição Genética para Doença , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Melanoma/genética , Mutação , Neoplasias/metabolismo , Proteínas Nucleares , Proteínas/metabolismo , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor , Raios Ultravioleta/efeitos adversos
13.
J Clin Pathol ; 56(7): 507-11, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12835295

RESUMO

BACKGROUND/AIMS: The inhibitor of growth gene 1 (ING1) is a modulator of cell cycle checkpoints, apoptosis, and cellular senescence. The most widely expressed ING1 isoform is p33(ING1b), which can modulate p53, a molecule that is frequently altered in breast cancer. Reduced ING1 mRNA expression has been observed in primary breast cancer expressing wild-type p53. METHODS: p33(ING1b), p53, oestrogen receptor (ER), and progesterone receptor (PgR) expression was studied in 86 primary invasive breast cancers using immunohistochemistry. RESULTS: Reduced nuclear expression of p33(ING1b) was found in cancer cells, both in intensity and the proportion of cells staining. This was associated with enhanced cytoplasmic p33(ING1b) expression in a proportion of cases. Analysis of several known biological factors indicated that high grade tumours were of larger size and more often negative for ER and PgR expression. However, larger tumours were more frequently p53 negative. These results provide evidence that p33(ING1b) alterations are associated with more poorly differentiated tumours. Positive correlations were found between nuclear p33(ING1b) expression and both ER and PgR expression. CONCLUSIONS: Optimum function of p53 is dependent on p33(ING1b) so that a reduction of nuclear p33(ING1b) expression, as seen in this series, would be predicted to compromise p53 function. This study showed that p33(ING1b) alterations were associated with more poorly differentiated tumours. Therefore, p33(ING1b) expression could be used as a marker of differentiation in invasive breast cancer. These results support the view that loss of p33(ING1b) may be an important molecular event in the differentiation and pathogenesis of invasive breast cancer.


Assuntos
Neoplasias da Mama/química , Carcinoma/química , Núcleo Celular/química , Proteínas de Neoplasias/análise , Proteínas/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Carcinoma/patologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica/métodos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Nucleares , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Proteínas Supressoras de Tumor
14.
J Clin Oncol ; 21(11): 2077-84, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12775732

RESUMO

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.


Assuntos
Biomarcadores Tumorais/análise , Técnicas Genéticas/normas , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Garantia da Qualidade dos Cuidados de Saúde , Biomarcadores Tumorais/genética , Southern Blotting , Cromossomos Humanos Par 1/genética , DNA de Neoplasias/análise , Erros de Diagnóstico/prevenção & controle , Erros de Diagnóstico/estatística & dados numéricos , Europa (Continente) , Humanos , Hibridização in Situ Fluorescente , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Ploidias , Reação em Cadeia da Polimerase , Controle de Qualidade , Padrões de Referência , Terminologia como Assunto
15.
Gene ; 307: 1-11, 2003 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-12706883

RESUMO

Amplification of the MYCN oncogene in neuroblastoma is associated with poor prognosis. The amplified unit of DNA can be up to 1 Mb in size and so could contain additional genes which affect tumour phenotype. The neuroblastoma amplified gene (NAG) gene was initially located 400 kb telomeric to MYCN at 2p24 and reported to be co-amplified in 5/8 (63%) cell lines and 9/13 (70%) tumours. The sequence of a 4.5 kb transcript was proposed from the analysis of overlapping cDNA clones. However, our Northern blot hybridisation experiments indicate that the main RNA species expressed in neuroblastoma is 7-8 kb in size. We describe for the first time the cloning and sequencing of the 7.3 kb transcript of the NAG gene together with its precise genomic location and full exon structure. The 5' end of the gene is located 30 kb telomeric to DDX1, with the two genes lying in opposite orientations. The 52 exons of the 7.3 kb transcript cover 420 kb of genomic DNA. In vitro translation studies confirmed the protein coding potential of the transcript. Co-amplification of the entire NAG gene with MYCN was found in 1/6 (17%) neuroblastoma cell lines and 10/50 (20%) primary tumours. Previous studies had measured co-amplification of only the 5' end of the gene, nearest to MYCN. In this study, co-amplification of the NAG gene was found to be significantly associated with low disease stage in MYCN-amplified tumours (P=0.0063).


Assuntos
Proteínas de Neoplasias/genética , Neuroblastoma/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Genes/genética , Humanos , Íntrons , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Células Tumorais Cultivadas
16.
BJU Int ; 91(1): 99-104, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12614260

RESUMO

OBJECTIVES: To investigate the matrix metalloproteinases (MMPs) 2 and 9 in bladder cancer cell lines stimulated with epidermal growth factor (EGF), and to investigate the presence of gelatinases in the urine of patients with bladder tumours, in relation to the stage and grade of tumour and the EGF receptor (EGFR) status. PATIENTS, SUBJECTS AND METHODS: Conditioned media from cultured tumour cells were analysed by zymography. Urine samples from 28 patients with transitional cell carcinoma and 12 normal volunteers were also analysed. Western blotting was used to verify the bands of gelatinolytic activity. The EGFR status of the tumours was assessed by immunohistochemistry. RESULTS: MMP9 was induced by EGF in the RT112 but not the RT4 bladder tumour cell line, whereas MMP2 production was unaffected by EGF. Gelatin zymography of urine samples from patients with bladder tumours showed high levels of MMP activity, with 78% positive for MMP9 and 28% positive for MMP2. The total gelatinolytic and MMP9 activity were significantly higher in patients with high-stage invasive tumours than in those with superficial tumours (P < 0.05), and were higher than in normal controls. Gelatinolytic activity at 130 and 200 kDa in urine was identified as MMP9 and MMP2. There was no significant relationship of urinary MMP9 activity to EGFR status of the tumour. CONCLUSION: EGF induces MMP9 but not MMP2 in bladder cells. Analysis of urinary gelatinases is a useful noninvasive technique and both total gelatinase and MMP9 activity are associated with high stages of bladder tumours.


Assuntos
Fator de Crescimento Epidérmico/fisiologia , Metaloproteinases da Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Bexiga Urinária/enzimologia , Western Blotting , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/urina , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/urina , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/urina
17.
J Clin Pathol ; 55(8): 596-601, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12147653

RESUMO

BACKGROUND/AIMS: p33(ING1b) is a tumour suppressor protein involved in growth control and apoptosis. Suppression of p33(ING1b) expression is associated with the loss of cellular growth control and immortalisation, whereas its overexpression causes cell cycle arrest. Moreover, normal p33(ING1b) expression is essential for optimal function of p53. Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood, accounting for one third of all childhood malignancies. A variety of cytogenetic abnormalities have been described but there is no single abnormality common to all cases. Deregulation of the TP53 pathway is a common genetic abnormality in human malignancies. However, TP53 mutations are uncommon in ALL. It is possible that alternative mechanisms of regulation of the TP53 apoptosis pathway, such as modulation of p33(ING1b) expression, may be important in ALL. The aim of this study was to assess the expression of p33(ING1b) in childhood ALL. METHODS: One hundred and forty five patients with childhood ALL were investigated in this immunohistochemical study of the expression of p33(ING1b). RESULTS: Loss of nuclear expression of p33(ING1b) was seen in 78% of cases. This was associated with increased cytoplasmic expression of the protein. Kaplan Meier survival analysis demonstrated a trend towards a better prognosis for patients with tumours that had lost nuclear p33(ING1b). CONCLUSION: These results suggest that the loss of nuclear p33(ING1b) expression may be an important molecular event in the pathogenesis of childhood ALL.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas/metabolismo , Adolescente , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Feminino , Seguimentos , Genes Supressores de Tumor , Humanos , Lactente , Recém-Nascido , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas Nucleares , Prognóstico , Taxa de Sobrevida , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor
18.
Biochim Biophys Acta ; 1587(2-3): 194-205, 2002 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12084461

RESUMO

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.


Assuntos
Fluoruracila/farmacologia , Timidilato Sintase/biossíntese , Animais , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Indução Enzimática/efeitos dos fármacos , Fluoruracila/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Técnicas In Vitro , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
19.
Br J Cancer ; 86(7): 1117-23, 2002 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-11953859

RESUMO

One hundred and one pre-treatment primary central primitive neuroectodermal tumours were analysed for the expression of p53 protein by immunohistochemistry using the monoclonal antibody DO-7. The staining intensity was classified into four groups: strong, medium, weak and negative and strong staining intensity was associated with the poorest survival. DNA sequencing of the p53 gene was performed in 28 cases representing all four staining groups. Mutations were found in only three of the strong staining tumours suggesting that DNA mutations were not common events and that in the majority of the tumours with over-expressed p53, the protein was likely to be wild-type. Results of immunohistochemistry showed a significantly positive relationship between the expression of p53 and Bax and Bcl-2 proteins, but not Waf-1. Multivariate analyses supported the prognostic value of p53 immunostaining in central primitive neuroectodermal tumours and also of age and gender of patients.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias do Sistema Nervoso Central/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Tumores Neuroectodérmicos Primitivos/genética , Proteína Supressora de Tumor p53/biossíntese , Adolescente , Adulto , Fatores Etários , Idade de Início , Anticorpos Monoclonais , Neoplasias do Sistema Nervoso Central/patologia , Cerebelo/química , Criança , Pré-Escolar , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Primers do DNA , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Tumores Neuroectodérmicos Primitivos/patologia , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Análise de Sequência de DNA , Fatores Sexuais , Análise de Sobrevida , Proteína X Associada a bcl-2
20.
Histopathology ; 40(4): 360-6, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11943021

RESUMO

AIMS: Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation-associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co-operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia. METHODS AND RESULTS: Sixty-seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma. CONCLUSIONS: Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with 'activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.


Assuntos
Melanócitos/patologia , Melanoma/patologia , Proteínas/metabolismo , Neoplasias Cutâneas/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ciclo Celular , Núcleo Celular/química , Citoplasma/química , Proteínas de Ligação a DNA , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Melanócitos/química , Melanoma/metabolismo , Pessoa de Meia-Idade , Proteínas Nucleares , Neoplasias Cutâneas/metabolismo , Proteínas Supressoras de Tumor
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