RESUMEN
BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell's response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel. METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches. RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8 representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2 cells show enhanced migratory ability. At 72 h after paclitaxel, MAD2 cells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2 MCF7 cell line after paclitaxel reflecting the observed increase in senescence. CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/efectos de los fármacos , Paclitaxel/farmacología , Proteínas Represoras/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Senescencia Celular/genética , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteínas Mad2 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Huso Acromático/efectos de los fármacos , Huso Acromático/genética , Huso Acromático/metabolismo , TransfecciónRESUMEN
Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.
Asunto(s)
Actinas/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Inmediatas-Precoces/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Actinas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Citoplasma/metabolismo , Embrión de Mamíferos , Fibroblastos/efectos de los fármacos , Humanos , Células Mesangiales/citología , Células Mesangiales/efectos de los fármacos , Ratones , Fosforilación , Transporte de Proteínas , Transducción de Señal/efectos de los fármacosRESUMEN
Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Mesangio Glomerular/citología , Proteínas Inmediatas-Precoces/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Actinas/química , Actinas/metabolismo , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Proteínas del Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Péptidos y Proteínas de Señalización Intracelular , Paxillin , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Forty-one patients with multiple myeloma were treated with a novel stem cell mobilisation regimen. The primary end points were adequate stem cell mobilising ability (>1% circulating CD34-positive cells) and collection (> or = 4 x 10(6) CD34-positive cells/kg), and safety. The secondary end point was activity against myeloma. The regimen (d-TEC) consisted of dexamethasone, paclitaxel 200 mg/m(2) i.v., etoposide 60 mg/kg i.v., cyclophosphamide 3 g/m(2) i.v., and G-CSF 5-10 microg/kg/day i.v. A total of 84 cycles were administered to these 41 individuals. Patient characteristics included a median age of 53 years, a median of five prior chemotherapy cycles, and a median interval of 10 months from diagnosis of myeloma to first cycle of d-TEC. Seventy-five percent of the patients had stage II or III disease, 50% had received carmustine and/or melphalan previously, and 25% had received prior radiation therapy. Eighty-eight percent of patients mobilised adequately after the first cycle of d-TEC and 91% mobilized adequately after the second cycle. An adequate number of stem cells were collected in 32 patients. Of the remaining nine patients, three mobilised, but stem cells were not collected, two mobilised but stem cell collection was < 4 x 10(6) CD34-positive cells/kg, three did not mobilise, and one died of disease progression. Major toxicities included pancytopenia, alopecia, fever and stomatitis. One patient died from multi-organ failure and progressive disease. Fifty percent of evaluable patients demonstrated a partial response and 28.6% of patients had a minor response. This novel dose-intense regimen was safe, capable of stem cell mobilisation and collection, even in heavily pre-treated patients, and active against the underlying myeloma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Dexametasona/uso terapéutico , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Movilización de Célula Madre Hematopoyética/métodos , Mieloma Múltiple/terapia , Adulto , Anciano , Antieméticos/uso terapéutico , Antígenos CD/sangre , Antígenos CD34/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Terapia Combinada , Ciclofosfamida/administración & dosificación , Dexametasona/administración & dosificación , Esquema de Medicación , Etopósido/administración & dosificación , Femenino , Humanos , Leucaféresis , Lorazepam/uso terapéutico , Masculino , Persona de Mediana Edad , Ondansetrón/uso terapéutico , Paclitaxel/administración & dosificación , Taxoides , Trasplante AutólogoRESUMEN
Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.
Asunto(s)
Apoptosis , Matriz Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Glándulas Mamarias Animales/citología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Femenino , Flavonoides/farmacología , Imidazoles/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/tratamiento farmacológico , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Busulfano/administración & dosificación , Busulfano/efectos adversos , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Etopósido/administración & dosificación , Etopósido/efectos adversos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Proyectos Piloto , Recurrencia , Inducción de Remisión , Trasplante AutólogoRESUMEN
During the past century various theories of mental functioning that rest on a deterministic view of man have gained dominance. However, a review of psychodynamic writing shows that there has remained a need for some concept of autonomy and inner direction, as represented by the concepts of ego autonomy and behavioral self-control. In fact, some studies of mental health and psychotherapy can be interpreted as supporting the existential view that the experience of "freedom" and "choice" is a genuine phenomenon. Although concepts of behavioral self-control may reconcile these contradictions, the author concludes that no theories of mental functioning have entirely dealt with the paradoxes of autonomy, inner direction, and choice.
Asunto(s)
Teoría Psicológica , Volición , Adaptación Psicológica , Conducta de Elección , Toma de Decisiones , Ego , Existencialismo , Humanos , Trastornos Mentales/terapia , Principios Morales , Teoría Psicoanalítica , Psicoterapia , Psicología del Esquizofrénico , Valores SocialesAsunto(s)
Amitriptilina/sangre , Trastorno Bipolar/prevención & control , Administración Oral , Adulto , Amitriptilina/administración & dosificación , Amitriptilina/efectos adversos , Trastorno Bipolar/tratamiento farmacológico , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Nortriptilina/sangre , Recurrencia , Factores de TiempoRESUMEN
The past decade has seen the emergence of popular "new psychotherapies" such as Gestalt Therapy, Transactional Analysis, and Primal Therapy, each with substantial followings. This paper puts forward a hypothesis about what these divergent systems may possess in common. Certain aspects of these therapy systems, in particular the induction of new patients and trainees, are examined in terms of Paul Tillich's concepts of the universal necessity of a rationale for courage. The significance of Tillich's analysis of courage for various historical movements is reviewed. In each system an element of unconditional commitment to a particular world view is required, as evidenced in the writings of the proponents of these systems, for the patient to be accepted into the particular system. These theoretically divergent systems possess, in common, a factor of an initial commitment to and induction into a collective belief system comparable to what Tillich describes as "the courage to be as a part".
Asunto(s)
Psicoterapia/métodos , Terapia Gestalt , Humanos , Filosofía , Autoimagen , Análisis TransaccionalRESUMEN
In a double-blind study, three depressed subjects received thyrotropin-releasing hormone (TRH) on three successive days, and one subject similarly received placebo; all subjects were then given ECT. Two of the patients given TRH responded to ECT. One patient's reaction is of special significance because of her response to ECT, diminished thyroid-stimulating hormone response to TRH, increased growth hormone and prolactin response to stress, and antidepressant effect of TRH. These findings raise the possibility that previous conflicting reports about TRH's antidepressant effects stem from the combined study of endocrinologically distinct depressive subgroups and strongly suggest that there may be a specific subgroup that is responsive to TRH.