RESUMO
Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs. Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells. Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 µM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states. Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.
RESUMO
BACKGROUND: Current guidelines favour the use of bleeding stents over balloon tamponade (BT) for refractory variceal bleeding (VB) from oesophageal varices. However, data on the efficacy and safety of self-expandable metal SX-ELLA Danis stents (SEMS) are limited. METHODS: Cirrhotic patients receiving SEMS for VB at four tertiary care centres were included in this retrospective multicentre study. Rates of failure-to-control bleeding (within 5 days) and bleeding-related mortality (6 weeks) were assessed. RESULTS: SEMS controlled VB in 79.4% (27/34) of patients. In the rest of patients, other rescue treatments including endoscopic band ligation (EBL, n = 3), SEMS renewed (n = 2) or Linton (n = 2) were applied; however, VB was only controlled in one patient. Early rebleeding within six weeks occurred in 17.6% (6/34) patients. Median SEMS dwell time was three (IQR:6) days. Overall n = 13/34 (38.2%) patients died with SEMS in situ. After SEMS removal, rebleeding and bleeding-related death occurred in n = 7 (35%) and n = 5 (14.7%) patients respectively. Only 32.4% (10/34) patients did not experience any rebleeding within six weeks after SEMS removal. Bleeding-related mortality was 47.1% (n = 16/34) and the median survival after SEMS placement was 2.1 months. Notably, no patient received an early transjugular intrahepatic portosystemic shunt (TIPS). The most common adverse events were stent dislocations (n = 13; 38.2%), while ulcers/necrosis of the oesophageal mucosa was seen in only four (11.8%) patients. CONCLUSION: SEMS controlled refractory VB in most patients. However, bleeding-related mortality remained high. While SEMS dislocations were frequent, ulcers/necrosis of the oesophagus was rare. Further studies should investigate whether the wider use of early TIPS reduces bleeding-related mortality after SEMS placement.
Assuntos
Varizes Esofágicas e Gástricas/terapia , Hemorragia Gastrointestinal/terapia , Cirrose Hepática/complicações , Stents/efeitos adversos , Adulto , Idoso , Áustria , Endoscopia Gastrointestinal , Varizes Esofágicas e Gástricas/etiologia , Varizes Esofágicas e Gástricas/mortalidade , Feminino , Hemorragia Gastrointestinal/etiologia , Hemorragia Gastrointestinal/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Derivação Portossistêmica Transjugular Intra-Hepática , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Even though trastuzumab is an effective therapy in early stage Her-2+ breast cancer, 40-50% of advanced Her-2+ breast cancer patients develop trastuzumab resistance. A potential resistance mechanism is aberrant downstream signal transmission due to loss of phosphatase and tensin homologue (PTEN). This study investigated the relationship between the expression of PTEN and trastuzumab response in Her-2 overexpressing metastatic breast cancer patients. METHODS: Between 2000 and 2007, 164 patients with Her-2+ metastatic breast cancer received trastuzumab-based therapy in our institution. We analyzed PTEN status by immunohistochemistry of 115 available tumor tissues and analyzed associations with other histopathological parameters, response rate, progression free survival (PFS) and overall survival (OS) with a median follow-up of 60 months. RESULTS: Eighty patients were PTEN positive (69.6%) and 35 patients PTEN negative (30.4%). We found a significant association of the expression of PTEN and p53 (p = 0.041), while there was no association with grading, hormone receptor status, IGFR or MIB. We found significantly more cases with progressive disease under trastuzumab-based therapy in patients with PTEN positive breast cancers (p = 0.018), while there was no significant correlation with PFS or OS. CONCLUSION: In Her-2-positive metastatic breast cancers, PTEN positivity was significantly associated with progressive disease, but not with PFS or OS.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Adulto JovemRESUMO
PURPOSE: This phase I study was performed to evaluate coagulation alterations during extracorporeal circulation (ECC) induced whole body hyperthermia (WBHT) in 12 patients with advanced soft tissue sarcomas. METHODS: To distinguish between effects of normothermic ECC and ECC-WBHT, blood samples were drawn at different time points: at baseline, after 30 min on normothermic ECC, at the end of the heating period, and 24 h and 7 days thereafter. Standard coagulation tests, coagulation factors, thrombelastography,platelets and reticulated platelets, liver enzymes, and scintigraphic platelet imaging were performed. RESULTS: Normothermic ECC resulted in coagulation alterations most likely due to systemic anticoagulation. Induction of hyperthermia caused thrombocytopenia, increased fibrin degradation products,prolonged clotting times, alteration in coagulation factors, and increased liver enzymes. The majority of these effects was most pronounced 24 h after ECC-WBHT. In addition, late liver sequestration of platelets was demonstrated in scintigraphic imaging at that time point. CONCLUSIONS: Temporal correlation between hemostatic alterations and elevation in liver enzymes leads to the assumption that liver impairment might play a crucial role in coagulation disturbances observed during ECC-WBHT and thereafter, thus strongly supported by liver sequestration of platelets.Therefore a close monitoring of hepatic derived coagulation alterations in patients undergoing extracorporeal whole body hypothermia is warranted.
Assuntos
Transtornos da Coagulação Sanguínea/etiologia , Coagulação Sanguínea , Oxigenação por Membrana Extracorpórea/efeitos adversos , Hipotermia Induzida/efeitos adversos , Falência Hepática/etiologia , Sarcoma/terapia , Neoplasias de Tecidos Moles/terapia , Adulto , Anticoagulantes/uso terapêutico , Áustria , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/diagnóstico , Testes de Coagulação Sanguínea , Feminino , Humanos , Falência Hepática/sangue , Falência Hepática/diagnóstico , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de Risco , Sarcoma/secundário , Neoplasias de Tecidos Moles/patologia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Despite patient selection based on ERBB2 overexpression, not all patients benefit from trastuzumab therapy. We have investigated whether a ERBB2 gene dosage effect might provoke increased biological aggressiveness and altered trastuzumab sensitivity. Absolute ERBB2 copy numbers ("CN") and ERBB2/centromer 17 ratios ("R") were measured by FISH analysis in tumors of 127 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer. CN and R were both significantly associated with shorter time to first metastasis (TTM) (CN: OR: 1.099, 95% CI: 1.042-1.159; R: OR: 1.211, 95% CI: 1.080-1.357) and longer PFS (CN: OR: 0.917, 95% CI: 0.867-0.969; R: OR: 0.840, 95% CI: 0.743-0.949) in a continuous variable Cox's regression model. Tumors with ERBB2/centromer 17 ratios of <2.2 had a significantly shorter TTM (p = 0.002) and significantly longer PFS (p = 0.003) than tumors with low-level (R: 2.2-6) and high-level amplification (R: >6). Interestingly, when ERBB2 copy numbers were analyzed, a significantly shorter TTM (p = 0.001) and longer PFS (p = 0.026) were observed in the group with high-level amplified CN (CN: >13), while no difference was observed between non- and low-level amplified CN. R, but not CN, was an independent predictor of complete (CR; OR: 1.685; 95% CI: 1.122-2.532) and partial (PR; OR: 1.704; 95% CI: 1.136-2.556) response in logistic regression analysis. CR (p = 0.016) rates were significantly higher in the high-level amplification group (R > 6), but no difference existed in response rates between non- and low-level amplified tumors in Chi-square tests. High-level ERBB2 amplification is associated with shorter TTM, but improved response to trastuzumab in metastatic breast cancer.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor ErbB-2/genética , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Amplificação de Genes , Dosagem de Genes , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Modelos de Riscos Proporcionais , Receptor ErbB-2/biossíntese , TrastuzumabRESUMO
Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKTmTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using KaplanMeier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/secundário , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Proteínas de Neoplasias/biossíntese , Receptor ErbB-2/análise , Receptor ErbB-3/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/classificação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Estudos de Coortes , Árvores de Decisões , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Estimativa de Kaplan-Meier , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Prognóstico , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-3/genética , Receptor ErbB-3/imunologia , Estudos Retrospectivos , Método Simples-Cego , Trastuzumab , Resultado do TratamentoRESUMO
Approximately half of all HER2/neu-overexpressing breast cancer patients do not respond to trastuzumab-containing therapy. Therefore, there remains an urgent and unmet clinical need for the development of predictive biomarkers for trastuzumab response. Recently, several lines of evidence have demonstrated that the inflammatory tumor microenvironment is a major contributor to therapy resistance in breast cancer. In order to explore the predictive value of inflammation in breast cancer patients, we measured the inflammatory biomarkers serum ferritin and C-reactive protein (CRP) in 66 patients immediately before undergoing trastuzumab-containing therapy and evaluated their progression-free and overall survival. The elevation in pre-treatment serum ferritin (>250 ng/ml) or CRP (>7.25 mg/l) was a significant predictor of reduced progression-free survival and shorter overall survival. When patients were stratified based on their serum ferritin and CRP levels, patients with elevation in both inflammatory biomarkers had a markedly poorer response to trastuzumab-containing therapy. Therefore, the elevation in inflammatory serum biomarkers may reflect a pathological state that decreases the clinical efficacy of this therapy. Anti-inflammatory drugs and life-style changes to decrease inflammation in cancer patients should be explored as possible strategies to sensitize patients to anti-cancer therapeutics.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores/sangue , Neoplasias da Mama/mortalidade , Ferritinas/sangue , Mediadores da Inflamação/sangue , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Proteína C-Reativa/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Inflamação/etiologia , Pessoa de Meia-Idade , Receptor ErbB-2/imunologia , Taxa de Sobrevida , TrastuzumabRESUMO
PURPOSE: This phase I study was performed to evaluate the feasibility and toxicity of a new method of extracorporeal perfusion-induced whole body hyperthermia (WBHT) in patients with advanced sarcoma avoiding the need of intubation and general anesthesia. METHODS: One double-lumen femoral venous access was inserted by Seldinger's technique to obtain WBHT (41.8°C for 120 minutes) via an extracorporeal circuit. No concomitant chemotherapy was applied. Up to 4 treatments of WBHT were performed under moderate sedation in 6 spontaneously breathing patients. Invasive hemodynamic monitoring was performed by use of a pulmonary artery catheter. RESULTS: After their first WBHT session, 2 patients were excluded from further treatment due to transient liver toxicity or catheter-related complication, so a total of 12 cycles remained for analyses. In all patients, conscious sedation resulted in sufficient spontaneous respiration without the need for mandatory ventilation. Median time to reach the target temperature was 84 minutes (range 60-142). Hemodynamic changes revealed the expected hyperdynamic state: heart rate, cardiac index, and stroke volume index significantly increased (p<0.05), whereas blood pressure and systemic and pulmonary vascular resistance index significantly decreased (p<0.05). A net fluid balance of 5822±1766 mL as well as norepinephrine (mean; 0.062 µg·kg¹·min⻹) were necessary to maintain the mean arterial blood pressure >60 mmHg. CONCLUSION: Our data demonstrate the feasibility of this method of extracorporeal WBHT without mandatory ventilation. Hemodynamic side effects in spontaneously breathing patients during perfusion-induced WBHT seem less severe than those observed in radiant heat WBHT.
Assuntos
Oxigenação por Membrana Extracorpórea , Hemodinâmica , Hipertermia Induzida , Pulmão/fisiopatologia , Oxigênio/metabolismo , Mecânica Respiratória , Sarcoma/terapia , Neoplasias de Tecidos Moles/terapia , Adulto , Áustria , Cateterismo de Swan-Ganz , Sedação Consciente , Oxigenação por Membrana Extracorpórea/efeitos adversos , Estudos de Viabilidade , Feminino , Veia Femoral , Humanos , Hipertermia Induzida/efeitos adversos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Sarcoma/metabolismo , Sarcoma/fisiopatologia , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/fisiopatologia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: p95HER2 is an NH(2)-terminally truncated form of HER2 that lacks the trastuzumab binding site and is therefore thought to confer resistance to trastuzumab treatment. In this report, we introduce a new antibody that has enabled the first direct quantitative measurement of p95HER2 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. We sought to show that quantitative p95HER2 levels would correlate with outcome in trastuzumab-treated HER2-positive metastatic breast cancer. EXPERIMENTAL DESIGN: The novel p95HER2 antibody used here was characterized for sensitivity, specificity, and selectivity over full-length HER2. Quantitative p95HER2 levels were measured in 93 metastatic breast tumors using a VeraTag FFPE assay to determine the correlation of p95HER2 levels with outcomes. RESULTS: Within a cohort of trastuzumab-treated metastatic breast cancer patients, high levels of p95HER2 were found to correlate with shorter progression-free survival [hazard ratio (HR), 1.9; P = 0.017] and overall survival (HR, 2.2; P = 0.012) in patients with tumors selected to be HER2 positive by the VeraTag HER2 assay. For those with tumors found to be fluorescence in situ hybridization positive, elevated p95HER2 correlated similarly with shorter progression-free survival (HR, 1.8; P = 0.022) and overall survival (HR, 2.2; P = 0.009). CONCLUSIONS: We have successfully generated an antibody that can specifically detect p95HER2, and developed an assay to quantify expression in FFPE tumor specimens. Using this novel assay, we have identified a group of HER2-positive patients expressing p95HER2 that have a worse outcome while on trastuzumab. As p95HER2 retains sensitivity to kinase inhibitors, measurement of p95HER2 in breast tumor sections may be useful in guiding treatment for patients with HER2-positive breast cancer.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Especificidade de Anticorpos , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Western Blotting , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Separação Celular , Estudos de Coortes , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Formaldeído , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Inclusão em Parafina , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/imunologia , Sensibilidade e Especificidade , Fixação de Tecidos , Trastuzumab , Resultado do TratamentoRESUMO
ERBB2 amplification and consecutive overexpression is a predictor for poor prognosis in breast cancer patients. In addition, incomplete resection of ERBB2-overexpressing tumors leads to increased proliferation of residual breast cancer cells. While the local release of cytokines is thought to be responsible for the malignant behavior of remaining tumor tissue, the exact mechanism is still unknown. We have analyzed epidermal growth factor receptor (EGFR), activated (p)EGFR, and activated (p)ERBB2 protein expression in ERBB2-overexpressing and in non-ERBB2-overexpressing tumors from patients who underwent breast surgery and consecutive re-excision for involved margins, and compared expression levels by immunohistochemistry. While overall ERBB2 protein expression in the initial and the re-excised sample were comparable, we observed an increase in pERBB2 in ductal carcinomas in situ in both, ERBB2-overexpressing (16/21 vs 24/24; P=0.018, chi(2) test) and non-ERBB2-overexpressing tumors (3/28 vs 5/12; P=0.025, chi(2) test). pERBB2 was not increased in invasive tumors, regardless on whether the samples had been taken from a ERBB2-overexpressing (9/25 vs 6/17; P=0.261, chi(2) test) or a non-ERBB2-overexpressing tumor (1/27 vs 0/8; P=0.581, chi(2) test). EGFR expression was only detected in 1/47 ERBB2-overexpressing primary tumors and 2/48 non-ERBB2-overexpressing tumors, and was undetectable in re-excised specimen. Taken together, we have demonstrated an increase in ERBB2 receptor activation in incompletely resected preinvasive breast cancer. We hypothesize that receptor phosphorylation is caused by growth factor stimulation in response to intraoperative tissue damage, and perioperative inhibition of specific cytokines could become a promising therapeutic strategy.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal/metabolismo , Neoplasia Residual/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma in Situ/patologia , Carcinoma in Situ/cirurgia , Carcinoma Ductal/patologia , Carcinoma Ductal/cirurgia , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasia Residual/patologia , Neuregulina-1/metabolismo , Fosforilação/fisiologiaRESUMO
Unique among the retroviruses, mouse mammary tumor virus (MMTV) carries, in addition to the usual long terminal repeat (LTR) promoter, another promoter, P2, which is located in the central part of the proviral U3 sequence, within the LTR open reading frame (ORF). Using an in vitro reporter system based on a sensitive luciferase expression assay, we investigated the regulation of the P2 promoter in the context of the Mtv-2 and Mtv-8 genomes. Irrespective of the genomic source, the activity of the P2 promoter is regulated by a downstream-located enhancer and an upstream-located negative regulatory element (NRE), the activity of which overrides the activator. During this study, we unexpectedly detected another independent neighboring promoter that we called P3. The novel P3 promoter does not seem to be controlled by any NRE but is influenced by the same enhancer that modulates the P2 promoter. The respective transcription starts of the two promoters located in this tight cluster are only 61 bases apart. The transcripts originating from this promoter complex carry the same first intron, which is bound by canonical splice donor and splice acceptor sites located in the LTR. One novel doubly spliced transcript carrying a 459-nucleotide-long ORF was detected in several MMTV-carrying murine cells and could be successfully expressed in murine cells as a His-tagged fusion product. The novel viral protein, the function of which remains to be elucidated, has an apparent molecular mass of 20 kDa.
Assuntos
Regulação Viral da Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes Reporter , Luciferases/análise , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Sítios de Splice de RNA , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.
Assuntos
Linhagem Celular , Cirrose Hepática Experimental/etiologia , Fígado/citologia , Proteína Supressora de Tumor p14ARF/deficiência , Actinas/metabolismo , Animais , Apoptose , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Movimento Celular , Inibidor p16 de Quinase Dependente de Ciclina , Desmina/metabolismo , Matriz Extracelular/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Camundongos , Pró-Colágeno/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
Immortalized p19(ARF) null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-beta driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-beta, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-beta mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-beta mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-beta mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-beta activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-beta to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade NeoplásicaRESUMO
Primary hepatocytes are blocked in mitotic activity and well-defined culture conditions only allow the limited expansion of these cells. Various genetic modifications have therefore been employed to establish immortalized hepatic cell lines, but, unfortunately, proper hepatocyte cultures conducting a faithful hepatic gene expression program and lacking malignancy are hardly available. Here we report the immortalization of primary hepatocytes isolated from p19(ARF) null mice using the rationale that loss of p19(ARF) lowers growth-suppressive functions of p53 and bypasses cellular senescence without losing genetic stability. The established hepatocytes, called MIM, express liver-specific markers, show a nontumorigenic phenotype, and competence to undergo Fas-mediated apoptosis. Intrasplenic transplantation of GFP-expressing parental MIM cells into Fas-injured livers of SCID mice revealed liver-reconstituting activity. In the regenerated liver, MIM cells localized in small-sized clusters and showed presence in structures comparable to canals of Hering, the site of oval cells. Transplantation of MIM-Bcl-X(L) cells, which are protected against apoptosis, and successive Fas-induced liver damage, enhanced donor-derived liver repopulation by showing differentiation into cholangiocytes and cells expressing markers characteristic of both fetal hepatocytes and oval cells. In conclusion, these data indicate that long-term cultivated p19(ARF) null hepatocytes are capable of generating hepatic progenitor cells during liver restoration, and thus represent a highly valuable tool to study the differentiation repertoire of hepatocytes.
Assuntos
Hepatócitos/metabolismo , Hepatócitos/transplante , Hepatopatias/cirurgia , Fígado/patologia , Células-Tronco/patologia , Proteína Supressora de Tumor p14ARF/deficiência , Animais , Divisão Celular , Linhagem Celular Transformada , Inibidor p16 de Quinase Dependente de Ciclina , Ducto Hepático Comum/patologia , Hepatócitos/patologia , Hepatopatias/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Baço/cirurgia , Transplante Heterotópico , Receptor fas/imunologiaRESUMO
Immune therapy for melanoma largely relies on preexisting T-cell responses. However, recent reports demonstrated the localized nature of such responses. Thus, we characterized the effect of immune therapy on the distribution of clonotypic T cells. To this end, we analyzed the T-cell receptor repertoire of multiple metastases of differentially treated melanoma patients revealing oligoclonal T-cell responses and the occurrence of identical T-cell clones in several metastases. However, these findings were not limited to immune therapy but were also observed after chemotherapy, suggesting its similar impact on the distribution of T cells.