RESUMO
Objective: To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) . Methods: Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients. Results: WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ(2)=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter. Conclusion: WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.
Assuntos
Síndromes Mielodisplásicas , Proteínas WT1/genética , Medula Óssea , Humanos , Síndromes Mielodisplásicas/genética , Prognóstico , RNA MensageiroRESUMO
Alanyl-glutamine (Ala-Gln), a highly soluble and stable glutamine dipeptide, is known to improve gut integrity and function. The aim of this study was to evaluate whether dietary Ala-Gln supplementation could improve growth performance, intestinal development and digestive-absorption function in weaned piglets. A total of 100 purebred Yorkshire piglets weaned at 21 days of age were assigned randomly to four dietary treatment groups and fed a basal diet (control group) or a basal diet containing 0.15%, 0.30% and 0.45% Ala-Gln, respectively. Compared with the control group, piglets fed the Ala-Gln diets had higher average daily gain and lower feed : gain and diarrhea rate (P < 0.05). Moreover, dietary Ala-Gln supplementation increased villous height and villous height : crypt depth ratio in duodenum and jejunum (P < 0.05), as well as the activities of maltase and lysozyme in jejunum mucosa (P < 0.05). In addition, a decrease in serum diamine oxidase activity and crypt depth in duodenum and jejunum was observed in piglets fed the Ala-Gln diets (P < 0.05). Serum cytosolic phospholipase A2 (cPLA2) concentration and gene expression of cPLA2, Na+-dependent glucose transporter 1, glucose transporter 2 and peptide transporter 1 in jejunum were increased by feeding Ala-Gln diets relative to control diet (P < 0.05). These results indicated that feeding Ala-Gln diet has beneficial effects on the growth performance of weaned piglets, which associated with maintaining intestinal morphology and digestive-absorption function.
Assuntos
Suplementos Nutricionais , Dipeptídeos/farmacologia , Suínos/crescimento & desenvolvimento , Animais , Dieta/veterinária , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/anatomia & histologia , Intestinos/efeitos dos fármacos , Masculino , Suínos/anatomia & histologia , Suínos/metabolismo , DesmameRESUMO
OBJECTIVE: To examine the expression of Galectin-3 and TRAIL in breast cancer tissue and their effects on the proliferation and apoptosis of breast cancer cells. PATIENTS AND METHODS: Breast cancer and normal adjacent tissue were collected from 120 patients pathologically diagnosed with breast cancer who underwent a modified radical mastectomy. SP method of immunohistochemistry was used to detect the expression levels of Galectin-3 and TRAIL in breast cancer tissues and normal adjacent tissues. The correlation between the expressions of Galectin-3 and TRAIL, and clinical prognosis of breast cancer were analyzed. Breast cancer cells were transfected with Galectin-3 siRNA and TRAIL overexpression constructs. Cell proliferation was measured by XTT method, and apoptosis was detected by flow cytometry. RESULTS: Higher Galectin-3 level and lower TRAIL level were found in breast cancer tissues compared with those in normal adjacent tissues (p < 0.001). High expression level of Galectin-3 and low expression level of TRAIL were found to be positively correlated with the shorter median survival time and overall survival time. Galectin-3 silencing by siRNA interference and TRAIL overexpression significantly decreased cell viability of MDA-MB-231 and increased the number of apoptotic cells. CONCLUSIONS: The expression level of Galectin-3 in breast cancer tissues was significantly increased compared with that in normal tissues, while the level of TRAIL protein was significantly decreased in cancer tissue. The biological role of these two proteins seems to be synergistic in inhibiting apoptosis of cancer cells. Therefore, the evaluation method that combined both Galectin-3 and TRAIL is of great clinical value in the evaluation of clinical prognosis of patients with breast cancer.
Assuntos
Neoplasias da Mama/patologia , Galectina 3/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Apoptose , Proteínas Sanguíneas , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Feminino , Galectina 3/análise , Galectinas , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/análiseRESUMO
The tyrosine kinase WEE1 controls the timing of entry into mitosis in eukaryotes and its genetic deletion leads to pre-implantation lethality in mice. Here, we show that besides the premature mitotic entry phenotype, Wee1 mutant murine cells fail to complete mitosis properly and exhibit several additional defects that contribute to the deregulation of mitosis, allowing mutant cells to progress through mitosis at the expense of genomic integrity. WEE1 interacts with the anaphase promoting complex, functioning as a negative regulator, and the deletion of Wee1 results in hyper-activation of this complex. Mammary specific knockout mice overcome the DNA damage response pathway triggered by the mis-coordination of the cell cycle in mammary epithelial cells and heterozygote mice spontaneously develop mammary tumors. Thus, WEE1 functions as a haploinsufficient tumor suppressor that coordinates distinct cell division events to allow correct segregation of genetic information into daughter cells and maintain genome integrity.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Glândulas Mamárias Animais/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Células Epiteliais/patologia , Feminino , Deleção de Genes , Instabilidade Genômica , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Mitose , TamoxifenoRESUMO
Recent studies have shown that induced pluripotent stem cells (iPSCs) retain a memory of their origin and exhibit biased differentiation potential. This finding reveals a severe limitation in the application of iPSCs to cell-based therapy because it means that certain cell types are not available for reprogramming for patients. Here we show that the iPSC differentiation process is accompanied by profound gene expression and epigenetic modifications that reflect cells' origins. Under typical conditions for mammary differentiation, iPSCs reprogrammed from tail-tip fibroblasts (TF-iPSCs) activated a fibroblast-specific signature that was not compatible with mammary differentiation. Strikingly, under optimized conditions, including coculture with iPSCs derived from the mammary epithelium or in the presence of pregnancy hormones, the fibroblast-specific signature of TF-iPSCs obtained during differentiation was erased and cells displayed a mammary-specific signature with a markedly enhanced ability for mammary differentiation. These findings provide new insights into the precise control of differentiation conditions that may have applications in personalized cell-based therapy.
Assuntos
Reprogramação Celular/genética , Epigênese Genética , Células Epiteliais/citologia , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Glândulas Mamárias Animais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Técnicas de Cocultura , Ilhas de CpG , Metilação de DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Estrogênios/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Nus , Gravidez , Progesterona/farmacologia , TranscriptomaRESUMO
Cancer metastasis is a lethal problem that claims the lives of over 90% of cancer patients. In this study, we have investigated metastatic potential of cancer stem cells (CSCs) isolated from mammary tumors of a Brca1-mutant mouse model. Our data indicated that CSCs, which are enriched in CD24(+)CD29(+)/CD49f(+) cell population, displayed much higher migration ability than CD24(-)CD29(-)/CD49f(-) cells in tissue culture and enhanced metastatic potential in allograft-nude mice. CD24(+)CD29(+) cells maintained the ability to differentiate and reconstitute heterogeneity in the metastatic tumors whereas CD24(-)CD29(-) cells could not. Corresponding to their enhanced metastatic ability, CD24(+)CD29(+) cells exhibited features of the epithelial to mesenchymal transition. Finally, using short hairpin RNA to knock down CD29 and/or CD49f in metastatic cancer cells, we demonstrated that while acute knockdown of CD29 or CD49f alone slightly decreased cell migration ability, knockdown of both genes generated a profound effect to block their migration, revealing an overlapping, yet critical function of both genes in the migration of CSCs. Our findings indicate that in addition to serving as markers of CSCs, CD29 and CD49f may also serve as potential therapeutic targets for cancer metastasis.
Assuntos
Proteína BRCA1/genética , Integrina alfa6/metabolismo , Neoplasias Mamárias Experimentais/patologia , Células-Tronco Neoplásicas/patologia , Receptores para Leptina/metabolismo , Animais , Biomarcadores Tumorais , Antígeno CD24/metabolismo , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Integrina alfa6/genética , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Camundongos Nus , Mutação , Células-Tronco Neoplásicas/metabolismo , Receptores para Leptina/genéticaRESUMO
Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection.
Assuntos
Microbolhas , Transfecção/métodos , Acústica , Animais , Aorta/citologia , Carbocianinas/química , Células Cultivadas , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Plasmídeos , RatosRESUMO
Our purpose was to demonstrate the use of radiofrequency spectral analysis to distinguish between benign and malignant lymph nodes with data obtained using electronic array echo endoscopes, as we have done previously using mechanical echo endoscopes. In a prospective study, images were obtained from eight patients with benign-appearing lymph nodes and 11 with malignant lymph nodes, as verified by fine-needle aspiration. Midband fit, slope, intercept, correlation coefficient, and root-mean-square (RMS) deviation from a linear regression of the calibrated power spectra were determined and compared between the groups. Significant differences were observable for mean midband fit, intercept, and RMS deviation (t test P < 0.05). For benign (n = 16) vs. malignant (n = 12) lymph nodes, midband fit and RMS deviation provided classification with 89 % accuracy and area under receiver operating characteristic (ROC) curve of 0.95 based on linear discriminant analysis. We concluded that the mean spectral parameters of the backscattered signals from electronic array echo endoscopy can provide a noninvasive method to quantitatively discriminate between benign and malignant lymph nodes.
Assuntos
Endossonografia/instrumentação , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Neoplasias/patologia , Área Sob a Curva , Endossonografia/métodos , Neoplasias Esofágicas/patologia , Análise de Fourier , Tumores do Estroma Gastrointestinal/secundário , Humanos , Neoplasias Pulmonares/patologia , Metástase Linfática , Linfoma/patologia , Neoplasias Pancreáticas/patologia , Curva ROC , Neoplasias Retais/patologia , Processamento de Sinais Assistido por ComputadorRESUMO
Expression of the breast cancer-associated gene 1 (BRCA1) in sporadic breast cancers is usually reduced, yet the underlying mechanisms remains elusive. To identify factors that are responsible for reduced BRCA1 expression, we screened 92 known transcription factors for their ability to regulate expression of BRCA1. Among several potential regulators, the Gli-Krueppel-related transcription factor Yin Yang 1 (YY1) showed the most dramatic transactivation of the BRCA1 promoter. YY1 binds to the promoter of BRCA1, and its overexpression resulted in increased expression of BRCA1 and a number of BRCA1 downstream genes. We further showed that overexpression of YY1 in cancer cells inhibited cell proliferation, foci formation and tumor growth in nude mice. To assess the clinical relevance between YY1 and BRCA1, we studied expression of YY1 and BRCA1 from human breast cancer samples and tissue arrays, and detected a significant positive correlation between the level of YY1 and BRCA1 expression in these cancers. Taken together, these findings suggest that YY1 is a key regulator of BRCA1 expression and may be causally linked to the molecular etiology of human breast cancer.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Genes BRCA1 , Proteínas Supressoras de Tumor/fisiologia , Fator de Transcrição YY1/fisiologia , Animais , Proteína BRCA1/análise , Mama/embriologia , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Ciclo Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Camundongos , Regiões Promotoras Genéticas , Fator de Transcrição YY1/análiseRESUMO
Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5MHz) ultrasound pulse (duration 13.3 or 40µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d=0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106±0.032µm (n=70) for acoustic pressure of 1.5MPa (duration 13.3µs), and increased to 0.171±0.030µm (n=125) for acoustic pressure of 1.7MPa and to 0.182±0.052µm (n=112) for a pulse duration of 40µs (1.5MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Membrana Celular/metabolismo , Microbolhas , Ultrassom/métodos , Acústica , Animais , Sobrevivência Celular/fisiologia , Feminino , Xenopus laevisRESUMO
Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. MSCs are involved in tissue repair and modulating immune responses in vitro and in vivo. From April 2005 to October 2008, 19 patients with refractory cGVHD were treated with MSCs derived from the BM of volunteers. The median dose of MSCs was 0.6 × 10(6) cells per kg body weight. Fourteen of 19 patients (73.7%) responded well to MSCs, achieving a CR (n=4) or a PR (n=10). The immunosuppressive agent could be tapered to less than 50% of the starting dose in 5 of 14 surviving patients, and five patients could discontinue immunosuppressive agents. The median duration between MSC administration and immunosuppressive therapy discontinuation was 324 days (range, 200-550 days). No patients experienced adverse events during or immediately after MSC infusion. The 2-year survival rate was 77.7% in this study. Clinical improvement was accompanied by the increasing ratio of CD5+CD19+/CD5-CD19+ B cells and CD8+CD28-/CD8+CD28+ T cells. In conclusion, transfusion of MSCs expanded in vitro, irrespective of the donor, might be a safe and effective salvage therapy for patients with steroid-resistant, cGVHD.
Assuntos
Doença Enxerto-Hospedeiro/cirurgia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Mesenquimais , Terapia de Salvação/métodos , Adolescente , Adulto , Doença Crônica , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunossupressores/administração & dosagem , Masculino , Resultado do Tratamento , Adulto JovemRESUMO
Checkpoint kinase 1 (Chk1) is a key element in the DNA-damage response pathway that is required for maintaining genomic stability. To study the potential role of Chk1 in mammary tumorigenesis, we disrupted it using a Cre/loxP system. We showed that although Chk1 heterozygosity caused abnormal development of the mammary gland, it was not sufficient to induce tumorigenesis. Simultaneous deletion of one copy of p53 failed to rescue the developmental defects; however, it synergistically induced mammary tumor formation in Chk1(+/-);MMTV-Cre animals with a median time to tumor latency of about 10 months. Chk1 deficiency caused a preponderance of abnormalities, including prolongation, multipolarity, misalignment, mitotic catastrophe and loss of spindle checkpoint, that are accompanied by reduced expression of several cell cycle regulators, including Mad2. On the other hand, we also showed that Chk1 deficiency inhibited mammary tumor formation in mice carrying a homozygous deletion of p53, uncovering a complex relationship between Chk1 and p53. Furthermore, inhibition of Chk1 with a specific inhibitor, SB-218078, or acute deletion of Chk1 using small hairpin RNA killed mammary tumor cells effectively. These data show that Chk1 is critical for maintaining genome integrity and serves as a double-edged sword for cancer: although its inhibition kills cancer cells, it also triggers tumorigenesis when favorable mutations are accumulated for cell growth.
Assuntos
Instabilidade Genômica , Neoplasias Mamárias Experimentais/genética , Proteínas Quinases/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Quinase 1 do Ponto de Checagem , Primers do DNA , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cariotipagem EspectralRESUMO
Mutations of SMAD4/DPC4 are found in about 60% of human invasive pancreatic ductal adenocarcinomas (PDACs); yet, the manner in which SMAD4 deficiency enhances tumorigenesis remains elusive. Using a Cre-LoxP approach, we generated a mutant mouse carrying a targeted deletion of Smad4 in the pancreas. We showed that the absence of Smad4 alone did not trigger pancreas tumor formation; however, it increased the expression of an inactivated form of Pten, suggesting a role of Pten in preventing Smad4-/- cells from undergoing malignancy. To investigate this, we disrupted both Pten and Smad4. We showed that Pten deficiency initiated widespread premalignant lesions, and a low tumor incidence that was significantly accelerated by Smad4-deficiency. The absence of Smad4 in a Pten-mutant background enhanced cell proliferation and triggered transdifferentiation from acinar, centroacinar and islet cells, accompanied by activation of Notch1 signaling. We showed that all tumors developed in the Smad4/Pten-mutant pancreas exhibited high levels of pAKT and mTOR, and that about 50 and 83% of human pancreatic cancers examined showed increased pAKT and pmTOR, respectively. Besides the similarity in gene expression, the pAKT and/or pmTOR-positive human PDACs and mouse pancreatic tumors also shared some histopathological similarities. These observations indicate that Smad4/Pten-mutant mice mimic the tumor progression of human pancreatic cancers that are driven by activation of the AKT-mTOR pathway, and uncovered a synergistic action of Smad4 and Pten in repressing pancreatic tumorigenesis.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad4/metabolismo , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Transdiferenciação Celular/fisiologia , Expressão Gênica , Genótipo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad4/genética , Serina-Treonina Quinases TORRESUMO
Ultrasound application in the presence of microbubbles is a promising strategy for intracellular drug and gene delivery, but it may also trigger other cellular responses. This study investigates the relationship between the change of cell membrane permeability generated by ultrasound-driven microbubbles and the changes in intracellular calcium concentration ([Ca(2+)](i)). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1MHz, 10-15cycles, 0.27MPa) in the presence of a Definity(TM) microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca(2+)](i) and dye loss from the cells were measured with preloaded fura-2. The ultrasound exposure generated fragmentation or shrinking of the microbubble. Only cells adjacent to the ultrasound-driven microbubble exhibited propidium iodide uptake with simultaneous [Ca(2+)](i) increase and fura-2 dye loss. The amount of PI uptake was correlated with the amount of fura-2 dye loss. Cells with delayed [Ca(2+)](i) transients from the time of ultrasound application had no uptake of PI. These results indicate the formation of non-specific pores in the cell membrane by ultrasound-stimulated microbubbles and the generation of calcium waves in surrounding cells without pores.
Assuntos
Cálcio/administração & dosagem , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos/métodos , Microbolhas , Ultrassom , Animais , Linhagem Celular , Sobrevivência Celular , Mioblastos Cardíacos/citologia , RatosRESUMO
To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca(2+)](i)) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison microbubbles. At extracellular [Ca(2+)](o) of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of [Ca(2+)](i) in time) with durations of 100-180 s. Immediate [Ca(2+)](i) transients after ultrasound application were induced by ultrasound-mediated microbubble-cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium [Ca(2+)](i) levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca(2+)](i) in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7-20 microm/s over a distance >200 microm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca(2+) for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison microbubbles can generate transient [Ca(2+)](i) changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane.
Assuntos
Cálcio/metabolismo , Sonicação/métodos , Albuminas , Animais , Células CHO , Sinalização do Cálcio/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Cricetinae , Cricetulus , Corantes Fluorescentes , Fluorocarbonos , Fura-2/análogos & derivados , Microbolhas , PorosidadeRESUMO
This study investigated the effects of ultrasound on the intracellular [Ca(2+)] of Chinese hamster ovary cells in the presence of albumin-encapsulated Optison microbubbles. Cells were exposed to 1 MHz ultrasound (tone burst of 0.2 s duration, 0.45 MPa peak pressure) while immersed in solution of 0.9 mM Ca(2+). Calcium imaging of the cells was performed using digital video fluorescence microscopy and Ca(2+)-indicator dye fura-2AM. Experimental evidence indicated that ultrasound caused a direct microbubble-cell interaction resulting in the breaking and eventual dissolution of the microbubble and concomitant permeabilization of the cells to Ca(2+). These cells exhibited a large influx of Ca(2+) over 3-4 s and did not return to their equilibrium levels. Subsequently, some cells exhibited one or more Ca(2+) oscillations with the onset of oscillations delayed by 10-80 s after the ultrasound pulse. A variety of oscillations were observed including decaying oscillations returning to the baseline value over 35-100 s, oscillations superimposed on a more gradual recovery over 150-200 s, and oscillations continued with increased amplitude caused by a second ultrasound tone burst. The delays in onset appeared to result from calcium waves that propagated across the cells after the application of the ultrasound pulse.
Assuntos
Sinalização do Cálcio/fisiologia , Animais , Fenômenos Biofísicos , Biofísica , Células CHO , Cricetinae , Cricetulus , Microbolhas/efeitos adversos , Microscopia de Fluorescência , Microscopia de Vídeo , Estresse Mecânico , Ultrassom/efeitos adversosRESUMO
Estrogen and its receptor alpha (ERalpha) have been implicated in the tissue-specific tumorigenesis associated with BRCA1 mutations. However, the majority of breast cancers developed in human BRCA1 mutation carriers are ERalpha-negative, challenging the link between BRCA1 and estrogen/ERalpha in breast cancer formation. Using a mouse model lacking the full-length form of BRCA1, here we show that ERalpha is highly expressed in the premalignant mammary gland and initiation stages of tumorigenesis, although its expression is gradually diminished during mammary tumor progression. We demonstrate that the absence of full-length BRCA1 increases sensitivity of cells to estrogen-induced extracellular signal-regulated kinase 1/2 phosphorylation and cyclin D1 expression. The absence of BRCA1 turns the proliferation of ERalpha-positive cells from a paracrine fashion to an autocrine or endocrine fashion. Consequently, BRCA1-mutant cells are sensitized to estrogen-induced cell proliferation in vitro and mammary tumorigenesis in vivo. These findings illustrate a molecular mechanism for estrogen/ERalpha signals in BRCA1-associated tissue-specific tumor formation, and identify several key elements in the estrogen/ERalpha-signaling cascade that can serve as potential therapeutic targets for BRCA1-associated tumorigenesis.
Assuntos
Proteína BRCA1/fisiologia , Estrogênios/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína BRCA1/genética , Linhagem Celular , Ciclina D1/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , FosforilaçãoRESUMO
The breast tumor associated gene-1 (BRCA1) and poly(ADP-ribose) polymerase-1 (PARP1) are both involved in DNA-damage response and DNA-damage repair. Recent investigations have suggested that inhibition of PARP1 represents a promising chemopreventive/therapeutic approach for specifically treating BRCA1- and BRCA2-associated breast cancer. However, studies in mouse models reveal that Parp1-null mutation results in genetic instability and mammary tumor formation, casting significant doubt on the safety of PARP1 inhibition as a therapy for the breast cancer. To study the genetic interactions between Brca1 and Parp1, we interbred mice carrying a heterozygous deletion of full-length Brca1 (Brca1(+/Delta11)) with Parp1-null mice. We show that Brca1(Delta11/Delta11);Parp1(-/-) embryos die before embryonic (E) day 6.5, whereas Brca1(Delta11/Delta11) embryos die after E12.5, indicating that absence of Parp1 dramatically accelerates lethality caused by Brca1 deficiency. Surprisingly, haploinsufficiency of Parp1 in Brca1(Delta11/Delta11) embryos induces a severe chromosome aberrations, centrosome amplification, and telomere dysfunction, leading to apoptosis and accelerated embryonic lethality. Notably, telomere shortening in Brca1(Delta11/Delta11);Parp1(+/-) MEFs was correlated with decreased expression of Ku70, which plays an important role in telomere maintenance. Thus, haploid loss of Parp1 is sufficient to induce lethality of Brca1-deficient cells, suggesting that partial inhibition of PARP1 may represent a practical chemopreventive/therapeutic approach for BRCA1-associated breast cancer.