RESUMO
Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.
Assuntos
Sistema Nervoso Central/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Anticorpos Neutralizantes/imunologia , Membrana Basal/metabolismo , Barreira Hematoencefálica/metabolismo , Medula Óssea , Movimento Celular , Sistema Nervoso Central/irrigação sanguínea , Sistema Nervoso Central/metabolismo , Líquido Cefalorraquidiano/metabolismo , Circulação Cerebrovascular , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Progressão da Doença , Feminino , Xenoenxertos/imunologia , Xenoenxertos/patologia , Integrina alfa6/imunologia , Integrina alfa6/metabolismo , Laminina/metabolismo , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Laminina/antagonistas & inibidores , Receptores de Laminina/imunologia , Receptores de Laminina/metabolismo , Crânio , Espaço SubaracnóideoRESUMO
A role for prolactin (PRL) in the pathogenesis of breast cancer has been confirmed at the cellular level in vitro, with multiple transgenic and knockout models in vivo, and within sizable patient populations through epidemiologic analysis. It is the obvious "next step" that these findings are translated into meaningful therapies to block PRL/PRLr function in human breast cancer. Several broad categories of PRL/PRLr antagonists are discussed in their pre-clinical context, including inhibitors of endocrine PRL elaboration, mutant ligand antagonists, ligand chimeras, and inhibitors of PRL-induced signaling and transactivation. The clinical potential for GHr antagonists are also discussed. These varied approaches all have demonstrated as proof-of-principle that PRL/PRLr antagonism can inhibit the in vitro and in vivo growth of breast cancer. Further pre-clinical development is required for most, however, before translation to clinical trials in breast cancer patients can occur.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Prolactina/antagonistas & inibidores , Prolactina/metabolismo , Animais , Antineoplásicos/imunologia , Neoplasias da Mama/imunologia , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Humanos , Imunoterapia , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/genética , Receptores da Prolactina/imunologia , Receptores da Prolactina/metabolismoRESUMO
Cells undergoing apoptosis are characterized by decreased cell size due to changes in intracellular ion concentration and rapid, aquaporin (AQP)-dependent water movement out of the cell, events required for the activation of pro-apoptotic enzymes. The current study demonstrates AQP 8 and 9 expression is significantly decreased in hepatocellular carcinoma (HCC) versus normal liver. Isolation of hepatic tumor cells (H4IIE) and hepatocytes confirmed a lack of water movement across the H4IIE cell membrane via AQPs and identified an inherent resistance of H4IIE cells to apoptotic stimuli. In contrast, hepatocytes rapidly responded to osmotic challenge through AQP-dependent water movement and underwent cell death following apoptotic stimulation.
Assuntos
Aquaporinas/metabolismo , Animais , Apoptose , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Células Cultivadas , Fígado/metabolismo , Neoplasias Hepáticas Experimentais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos ACI , Água/metabolismoRESUMO
Interlukin-6 (IL-6) is a pleitropic cytokine that plays a central role in normal and abnormal hepatic function and response. The aims of the current study were to determine the viability of using cell encapsulation technology to introduce a genetically modified xenogeneic (CHO) cell population to elevate circulating levels of rhIL-6 in a rat model and determine the effects of sustained high rhIL-6 levels on hepatocellular carcinoma (HCC) progression in vivo. An alginate matrix was combined with transfected CHO cells, selected for their ability to synthesize rhIL-6, and used to generate uniform alginate-cell beads. Once encapsulated transfected cells continued to undergo replication, formed colonies within the bead, and synthesized/released large quantities of rhIL-6 into culture medium in vitro. Intraperitoneal implantation of beads into rats resulted in significantly increased circulating and intrahepatic levels of rhIL-6 up to 4 days postimplantation. Prolonged implantation led to the escape of CHO cells from the bead, resulting in a host response and CHO cell death within the bead. Subsequently CHO-IL-6 encapsulated cells were implanted into rats previously inoculated intrahepatically with the H4IIE HCC cell line. These studies demonstrated the maintenance of high circulating/intrahepatic rhIL-6 levels in this model. Despite significantly increased rhIL-6, this technique did not significantly alter the rate of net tumor progression. However, Stat3 activity was significantly increased in both normal liver and HCC tissue resected from animals implanted with CHO-IL-6 cells. Collectively these data demonstrate the short-term viability of using cell encapsulation technology to generate high levels of active circulating and intrahepatic cytokines and raise the possibility of modifying specific signal transduction cascades identified to be important during tumor progression.
Assuntos
Carcinoma Hepatocelular/terapia , Implantes de Medicamento , Interleucina-6/genética , Neoplasias Hepáticas Experimentais/terapia , Alginatos/química , Animais , Células CHO , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Cricetinae , Cricetulus , Progressão da Doença , Composição de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Interleucina-6/metabolismo , Interleucina-6/fisiologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Ratos , TransfecçãoAssuntos
Apoptose , Aquaporinas/metabolismo , Tamanho Celular , Água/fisiologia , Animais , Bioensaio , Transporte Biológico , Separação Celular , Feminino , Permeabilidade , Ratos , Ratos Sprague-Dawley , Timo/citologiaRESUMO
The host tissue microenvironment influences malignant cell proliferation and metastasis, but little is known about how tumor-induced changes in the microenvironment affect benign cellular ecosystems. Applying dynamic in vivo imaging to a mouse model, we show that leukemic cell growth disrupts normal hematopoietic progenitor cell (HPC) bone marrow niches and creates abnormal microenvironments that sequester transplanted human CD34+ (HPC-enriched) cells. CD34+ cells in leukemic mice declined in number over time and failed to mobilize into the peripheral circulation in response to cytokine stimulation. Neutralization of stem cell factor (SCF) secreted by leukemic cells inhibited CD34+ cell migration into malignant niches, normalized CD34+ cell numbers, and restored CD34+ cell mobilization in leukemic mice. These data suggest that the tumor microenvironment causes HPC dysfunction by usurping normal HPC niches and that therapeutic inhibition of HPC interaction with tumor niches may help maintain normal progenitor cell function in the setting of malignancy.
Assuntos
Medula Óssea/patologia , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Nicho de Células-Tronco/patologia , Animais , Antígenos CD34/análise , Benzilaminas , Contagem de Células , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CXCL12/metabolismo , Ciclamos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/fisiopatologia , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Nicho de Células-Tronco/fisiopatologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.
Assuntos
Endocitose/fisiologia , Janus Quinase 2/metabolismo , Prolactina/fisiologia , Receptores da Prolactina/metabolismo , Ubiquitinação/fisiologia , Catálise , Linhagem Celular , Endocitose/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Janus Quinase 2/deficiência , Ligantes , Fosforilação/efeitos dos fármacos , Prolactina/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacosRESUMO
A member of the family of hematopoietic cytokines, human prolactin (hPRL) serves a dual role both as an endocrine hormone and as an autocrine/paracrine cytokine or growth factor. During investigation of the solution structural properties of hPRL, we have noted a surprising pH dependence of its structural stability over a range from approximately pH 6.0 to pH 8.0. An analysis of backbone atom NMR chemical shift changes and backbone amide hydrogen-deuterium exchange rates due to titration of the solution pH over this same range, along with calculations of protein surface electrostatic potential, suggests the possible involvement of a localized cluster of three His residues (27, 30, and 180), which comprise a portion of the high-affinity receptor-binding epitope. Surface plasmon resonance analysis of the interaction between hPRL and the extracellular domain (ECD) of the hPRL receptor reveals a selective 500-fold change in the dissociation rate between pH 8.3 and pH 5.8. In comparison, the interaction of hGH with the same receptor ECD did not demonstrate any significant dependence on pH. We also present an initial investigation of the pH dependence of hPRL function in rat Nb2 cell proliferation assays and a STAT5 luciferase gene reporter assay in the T47D human breast cancer cell line, whose results are consistent with our biophysical studies. The potential implications of this variation in hPRL's structural stability and receptor-binding kinetics over this physiologic range of pH are discussed.
Assuntos
Hormônio do Crescimento/metabolismo , Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Prolactina/química , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
BACKGROUND INFORMATION: During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. RESULTS: In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. CONCLUSIONS: Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.
Assuntos
Apoptose , Aquaporina 1/metabolismo , Aquaporinas/metabolismo , Caveolina 1/fisiologia , Animais , Aquaporina 1/química , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Ratos , Água/metabolismoRESUMO
Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.
Assuntos
Aquaporinas/fisiologia , Água Corporal/fisiologia , Estrogênios/fisiologia , Útero/fisiologia , Algoritmos , Animais , Western Blotting , Permeabilidade da Membrana Celular/fisiologia , Tamanho Celular , Células Epiteliais/metabolismo , Estradiol/biossíntese , Feminino , Imunofluorescência , Camundongos , Ovariectomia , Testes de Precipitina , Progesterona/biossíntese , Útero/citologia , Útero/metabolismo , ViscosidadeRESUMO
Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C(6)-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K(+) from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K(+) concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K(+) concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane.